Butyrylcholinesterase variants and methods of use

ABSTRACT

The invention provides four butyrylcholinesterase variants having increased cocaine hydrolysis activity as well as the corresponding encoding nucleic acids. The invention also provides libraries comprising butyrylcholinesterase variants having at least one amino acid alteration in one or more regions of butyrylcholinesterase and further having at least one butyrylcholinesterase variant exhibiting enhanced cocaine hydrolysis activity compared to butyrylcholinesterase. The invention further provides methods of hydrolyzing a cocaine-based butyrylcholinesterase substrate as well as methods of treating a cocaine-induced condition.

This invention was made with government support under grant number 1R01DA011707 awarded by the National Institutes of Health. The United StatesGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

This invention relates to butyrylcholinesterase variants and, morespecifically to the production and therapeutic use thereof.

Cocaine abuse is a significant social and medical problem in the UnitedStates as evidenced by the estimated 3.6 million chronic users. Cocaineabuse often leads to long-term dependency as well as life-threateningoverdoses. However, no effective antagonist is currently available thatcombats the reinforcing and toxic effects of cocaine.

One difficulty in identifying an antagonist to treat cocaine abusearises largely from the narcotic's mechanism of action. Specifically,cocaine inhibits the re-uptake of neurotransmitters resulting inover-stimulation of the reward pathway. It is this over-stimulation thatis proposed to be the basis of cocaine's reinforcing effect. Inaddition, at higher concentrations, cocaine interacts with multiplereceptors in both the central nervous and cardiovascular systems,leading to toxicities associated with overdose. Because of thismultifarious mechanism of action of cocaine, it is difficult to identifyselective antagonists to treat both the reinforcing and toxic effects ofcocaine. Additionally, antagonists that block cocaine's binding to itsreceptors tend to display many of the same deleterious effects ascocaine.

Recently, alternative treatment strategies based on intercepting andneutralizing cocaine in the bloodstream have been proposed. For example,dopamine D1, D2, and D3 antagonists affect the reinforcing potency ofcocaine in the rat model, these antagonists display a narrow range ofeffective doses and the extent of decrease in cocaine potency is quitesmall. In addition, these dopamine antagonists produce profounddecreases in other behaviors when the doses are increased only slightlyabove the levels that display an effect on cocaine self-administrationbehavior.

A separate treatment strategy involves partial protection against theeffects of cocaine using antibody-based approaches. Limitations ofimmunization approaches include the stoichiometric depletion of theantibody following the binding of cocaine. The use of a catalyticantibody, which metabolizes cocaine in the bloodstream, partiallymitigates this problem by degrading and releasing cocaine, permittingbinding of additional cocaine. However, the best catalytic antibodyidentified to date metabolizes cocaine significantly slower thanendogenous human serum esterases.

In vivo, cocaine is metabolized by three principal routes: 1)N-demethylation in the liver to form norcocaine, 2) hydrolysis by serumand liver esterases to form ecgonine methyl ester, and 3) nonenzymatichydrolysis to form benzoylecgonine. In humans, norcocaine is a minormetabolite, while benzoylecgonine and ecgonine methyl ester account forabout 90% of a given dose. The metabolites of cocaine are rapidlycleared and appear not to display the toxic or reinforcing effects ofcocaine. Low serum levels of butyrylcholinesterase have been correlatedwith adverse physiological events following cocaine overdose, providingfurther evidence that butyrylcholinesterase accounts for the cocainehydrolysis activity observed in plasma. Human plasma obtained fromindividuals with a defective version of the butyrylcholinesterase genehas been shown to have little or no ability to hydrolyze cocaine invitro, and the hydrolysis of cocaine in plasma of individuals carryingone defective and one wild type copy of the butyrylcholinesterase genehas been shown to proceed at one-half the normal rate. Therefore, it hasbeen suggested that individuals with defective versions of thebutyrylcholinesterase gene are at higher risk for life-threateningreactions to cocaine. Recently, administration of butyrylcholinesterasehas been demonstrated to confer limited protection against cocaineoverdose in mice and rats.

Although administration of butyrylcholinesterase provides some effectagainst cocaine toxicity in vivo, it is not an efficient catalyst ofcocaine hydrolysis. The low cocaine hydrolysis activity of wild-typebutyrylcholinesterase requires the use of prohibitively large quantitiesof purified enzyme for therapy.

A number of naturally occurring human butyrylcholinesterases as well asspecies variations are known, none of which exhibits increased cocainehydrolysis activity. Similarly, although a variety of recombinantlyprepared butyrylcholinesterase mutations have been tested for increasedcocaine hydrolysis activity, only one such mutant, termed A328Y, hasbeen identified that exhibits increased cocaine hydrolysis activity.Further butyrylcholinesterase mutations that lead to increased cocainehydrolysis activity need to be identified to permit clinical evaluationof butyrylcholinesterase.

Thus, there exists a need for butyrylcholinesterase variants capable ofhydrolyzing cocaine significantly more efficiently than wild-typebutyrylcholinesterase. The present invention satisfies this need andprovides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides four butyrylcholinesterase variants havingincreased cocaine hydrolysis activity as well as the correspondingencoding nucleic acids. The invention also provides libraries comprisingbutyrylcholinesterase variants having at least one amino acid alterationin one or more regions of butyrylcholinesterase and further having atleast one butyrylcholinesterase variant exhibiting enhanced cocainehydrolysis activity compared to butyrylcholinesterase. The inventionfurther provides methods of hydrolyzing a cocaine-basedbutyrylcholinesterase substrate as well as methods of treating acocaine-induced condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A–1D shows the nucleic acid sequence designated SEQ ID NO: 1 andthe deduced amino acid sequence of the butyrylcholinesterase variantdesignated SEQ ID NO: 2.

FIG. 2 shows the amino acid sequence of human butyrylcholinesterase withthe seven regions designated SEQ ID NOS: 9 through 15 underlined andaromatic active gorge residues shaded: W82, W112, Y128, W231, F329,Y332, W430 and Y440.

FIG. 3 shows the nucleic acid sequence of human butyrylcholinesterase(SEQ ID NO: 16).

FIG. 4 shows an amino acid sequence alignment of human wild-type (SEQ IDNO: 17), human A variant (SEQ ID NO: 18), human J variant (SEQ ID NO:19), human K variant (SEQ ID NO: 20), horse (SEQ ID NO: 21), cat (SEQ IDNO: 22) and rat butyrylcholinesterase variants (SEQ ID NO: 23).

FIG. 5 shows (A) the correlation between the HPLC assay and the isotopetracer assay as demonstrated by plotting the quantitation of benzoicacid formation by both methods, and (B) the K_(m) for cocaine hydrolysisactivity of horse butyrylcholinesterase using the Lineweaver-Burkdouble-reciprocal plot.

FIG. 6 shows solid phase immobilization of wild-type (filled circles)and truncated (open circles) butyrylcholinesterase for measuring cocainehydrolysis activity.

FIG. 7 shows the use of multiple synthesis columns and codon-basedmutagenesis for the synthesis of focused libraries.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides four butyrylcholinesterase variants that exhibitincreased cocaine hydrolysis activity compared to butyrylcholinesterase.The identification of butyrylcholinesterase variants that exhibitincreased cocaine hydrolysis activity provides treatment options forcocaine-induced conditions such as cocaine overdose and cocaineaddiction.

In one embodiment, the invention provides a method of treating anindividual suffering from symptoms due to cocaine toxicity includinggrand-mal seizures, cardiac arrest, stroke, and drug-induced psychosisaccompanied by elevated blood pressure. The butyrylcholinesterasevariants of the invention hold significant clinical value because oftheir capability to hydrolyze cocaine at a higher rate than any of theknown naturally occurring variants. It is this increase in cocainehydrolysis activity that enables a much more rapid response to thelife-threatening symptoms of cocaine toxicity that sets thebutyrylcholinesterase variants of the invention apart from othertreatment options.

The invention also provides libraries of butyrylcholinesterase variantsas well as of nucleic acids encoding butyrylcholinesterase variants. Thebutyrylcholinesterase variant libraries of the invention have one ormore amino acid alterations in regions determined to be important forcocaine hydrolysis activity. Therefore, the invention provides librariesthat can be screened for butyrylcholinesterase variants exhibitingincreased cocaine hydrolysis activity.

As used herein, the term “butyrylcholinesterase” is intended to refer toa polypeptide having the sequence of naturally occurringbutyrylcholinesterase. A naturally occurring butyrylcholinesterase canbe of any species origin, for example, human, primate, horse, or murine.Therefore, a butyrylcholinesterase can be, for example a mammalianbutyrylcholinesterase. In addition, a butyrylcholinesterase of theinvention can be an isotype variation, polymorphism or any other allelicvariation of a naturally occurring butyrylcholinesterase. A nucleic acidencoding a butyrylcholinesterase of the invention encodes a polypeptidehaving the sequence of any naturally occurring butyrylcholinesterase.Therefore, a nucleic acid encoding a butyrylcholinesterase can encode abutyrylcholinesterase of any species origin, for example, human,primate, horse, or murine. In addition, a nucleic acid encoding abutyrylcholinesterase encompasses any naturally occurring allele,isotype or polymorphism.

As used herein, the term “butyrylcholinesterase variant” is intended torefer to a molecule that is structurally similar tobutyrylcholinesterase, but differs by at least one amino acid frombutyrylcholinesterase. A butyrylcholinesterase variant has substantiallythe same amino acid sequence as butyrylcholinesterase and exhibitscocaine hydrolysis activity. In this regard, a butyrylcholinesterasevariant can possess, for example, reduced, substantially the same orincreased cocaine hydrolysis activity compared to butyrylcholinesterase.For example, the cocaine hydrolysis activity of a butyrylcholinesterasevariant of the invention can be increased by a factor of 5, 10, 50, 100or more.

A butyrylcholinesterase variant can have a single amino acid alterationas well as multiple amino acid alterations compared tobutyrylcholinesterase. A specific example of a butyrylcholinesterasevariant is butyrylcholinesterase having the amino acid Tryptophan atposition 328, of which the amino acid sequence and encoding nucleic acidsequence is shown in FIG. 1 and designated as SEQ ID NOS: 2 and 1,respectively. Additional examples of butyrylcholinesterase variants arebutyrylcholinesttrase having the amino acid Glycine at position 287, ofwhich the amino acid sequence and nucleic acid sequence are describedherein and designated SEQ ID NOS: 4 and 3, respectively;butyrylcholinesterase having the amino acid Glutamine at position 285,of which the amino acid sequence and nucleic acid sequence are describedherein and designated SEQ ID NOS: 6 and 5, respectively; andbutyrylcholinesterase having the amino acid Serine at position 285, ofwhich the amino acid sequence and nucleic acid sequence are describedherein and designated SEQ ID NOS: 8 and 7, respectively. The term isalso intended to include butyrylcholinesterase variants encompassing,for example, modified forms of naturally occurring amino acids such asD-stereoisomers, non-naturally occurring amino acids, amino acidanalogues and mimetics so long as such variants have substantially thesame amino acid sequence as butyrylcholinesterase and exhibit cocainehydrolysis activity. A butyrylcholinesterase variant of the inventioncan have one or more amino acid alterations outside of the regionsdetermined or predicted to be important for cocaine hydrolysis activityherein. Furthennore, a butyrylcholinesterase variant of the inventioncan have one or more additional modifications that do not significantlychange its cocaine hydrolysis activity. A butyrylcholinesterase variantof the invention can also have increased stability compared tobutyrylcholinesterase.

As used herein, the term “substantially the same” when used in referenceto an amino acid sequence is intended to mean a polypeptide, fragment orsegment having an identical amino acid sequence, or a polypeptide,fragment or segment having a similar, non-identical sequence that isconsidered by those skilled in the art to be a functionally equivalentamino acid sequence. An amino acid sequence that is substantiallyidentical to a reference butyrylcholinesterase or butyrylcholinesterasevariant of the invention can have at least 70%, at least 80%, at least81%, at least 83%, at least 85%, at least 90%, at least 95% or moreidentity to the reference butyrylcholinesterase. Substantially the sameamino acid sequence is also intended to include polypeptidesencompassing, for example, modified forms of naturally occurring aminoacids such as D-stereoisomers, non-naturally occurring amino acids,amino acid analogues and mimetics so long as such polypeptides retainfunctional activity as defined above. A biological activity of abutyrylcholinesterase variant of the invention is cocaine hydrolysisactivity as described herein. For example, the butyrylcholinesterasevariant A328W designated SEQ ID NO: 2 exhibits at least a fifteen-foldincreased cocaine hydrolysis activity compared to butyrylcholinesterase;the butyrylcholinesterase variant S287G designated SEQ ID NO: 4 exhibitsat least a four-fold increased cocaine hydrolysis activity compared tobutyrylcholinesterase; the butyrylcholinesterase variant P285Qdesignated SEQ ID NO: 6 exhibits approximately a four-fold increasedcocaine hydrolysis activity compared to butyrylcholinesterase; thebutyrylcholinesterase variant P285S designated SEQ ID NO: 8 exhibitsapproximately a three-fold increased cocaine hydrolysis activitycompared to butyrylcholinesterase.

It is understood that minor modifications in the primary amino acidsequence can result in a polypeptide that has a substantially equivalentfunction as compared to a polypeptide of the invention. Thesemodifications can be deliberate, as through site-directed mutagenesis,or may be accidental such as through spontaneous mutation. For example,it is understood that only a portion of the entire primary structure ofa butyrylcholinesterase variant can be required in order to effectcocaine hydrolysis activity. Moreover, fragments of the sequence of abutyrylcholinesterase variant of the invention are similarly includedwithin the definition as long as at least one biological function of thebutyrylcholinesterase variant is retained. It is understood that variousmolecules can be attached to a polypeptide of the invention, forexample, other polypeptides, carbohydrates, lipids, or chemicalmoieties.

As used herein, the term “corresponding to” refers to an amino acidsequence that is substantially the same as a reference amino acidsequence. The amino acid sequence can occupy the same or different aminoacid positions relative to the reference polypeptide, fragment orsegment. It is understood that, while butyrylcholinesterases ofdifferent species origin as well as allelic variations will havesubstantiaUy identical amino acid sequences, the physical locations aswell as the size of a particular amino acid sequence may vary.Therefore, the amino acids making up a given segment in abutyrylcholinesterase or butyrylcholinesterase variant may not be in thesame physical location or occupy the identical amino acid positions asin the reference butyrylcholinesterase or butyrylcholinesterase variant.For example, butyrylcholinesterases of different species origin as wellas allelic variations have substantially similar amino acid sequences,but the amino acid positions making up a region may not correspond tothose recited for SEQ ID NOS: 9 through 15. For example, a region thatis substantially similar in amino acid sequence to the region designatedas SEQ ID NO: 9 may not occupy amino acid positions 68–82 in a non-humanbutyrylcholinesterase or an allelic variation of any species origin, butis nevertheless encompassed by the present invention.

As used herein, the term “substantially the same” in reference to anucleic acid molecule of the invention or a fragment thereof includessequences having one or more additions, deletions or substitutions withrespect to the reference sequence, so long as the nucleic acid moleculeretains ita ability to selectively hybridize with the subject nucleicadd molecule under moderately stringent conditions, or highly stringentconditions. The term “moderately stringent conditions,” as used herein,refers to hybridization conditions equivalent to hybridization offilter-bound nucleic acid in 50% formamide, 5× Denhsrdt's solution,5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 02% SDS, at50°. As used herein, “highly stringent conditions” are conditionsequivalent to hybridization of filter-bound nucleic acid in 50%formamide, 5× Denherdt's solution, 5×SSPE, 0.2% SDS at 42° C., followedby washing in 0.2×SSPE, 0.2% SDS, at 65°. Other suitable moderatelystringent and highly stringent hybridization buffers and conditions arewell known to those of skill in the art and are described, for example,in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1992) and in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1998). Thus, it is not necessary that two nucleic, acids exhibitsequence identity to be substantially complementary, only that they canspecifically hybridize or be made to specifically hybridize withoutdetectable cross reactivity with other similar sequences.

In general, a nucleic acid molecule that has “substantially the same”nucleotide sequence as a reference sequence will have greater than about60% identity, such as greater than about 65%, 70%, 75% identity with thereference sequence, such as greater than about 80%, 85%, 90%, 95%, 97%or 99% identity to the reference sequence over the length of the twosequences being compared. Identity of any two nucleic acid sequences canbe determined by those skilled in the art based, for example, on a BLAST2.0 computer alignment, using default parameters. BLAST 2.0 searching isavailable at ncbi.nlm.nih.gov/gorf/bl2.html., as described by Tatiana etal., FEMS Microbiol Lett. 174:247–250 (1999).

As used herein, the term “fragment” when used in reference to a nucleicacid encoding the claimed polypeptides is intended to mean a nucleicacid having substantially the same sequence as a portion of a nucleicacid encoding a polypeptide of the invention or segments thereof. Thenucleic acid fragment is sufficient in length and sequence toselectively hybridize to a butyrylcholinesterase variant encodingnucleic acid or a nucleotide sequence that is complementary to abutyrylcholinesterase variant encoding nucleic acid. Therefore, fragmentis intended to include primers for sequencing and polymerase chainreaction (PCR) as well as probes for nucleic acid blot or solutionhybridization.

Similarly, the term “functional fragment” when used in reference to anucleic acid encoding a butyrylcholinesterase or butyrylcholinesterasevariant is intended to refer to a portion of the nucleic acid thatencodes a portion of the butyrylcholinesterase or butyrylcholinesterasevariant that still retains some or all of the cocaine hydrolysisactivity of the parent polypeptide. A functional fragment of apolypeptide of the invention exhibiting a functional activity can have,for example, at least 6 contiguous amino acid residues from thepolypeptide, at least 8, 10, 15, 20, 30 or 40 amino acids, and often hasat least 50, 75, 100, 200, 300, 400 or more amino acids of a polypeptideof the invention, up to the full length polypeptide minus one aminoacid.

As used herein, the term “functional fragment” in regard to apolypeptide of the invention, refers to a portion of the referencepolypeptide that is capable of exhibiting or carrying out a “functionalactivity” of the reference polypeptide. A functional fragment of apolypeptide of the invention exhibiting a functional activity can have,for example, at least 6 contiguous amino acid residues from thepolypeptide, at least 8, 10, 15, 20, 30 or 40 amino acids, and often hasat least 50, 75, 100, 200, 300, 400 or more amino acids of a polypeptideof the invention, up to the full length polypeptide minus one aminoacid. The appropriate length and amino acid sequence of a functionalfragment of a polypeptide of the invention can be determined by thoseskilled in the art, depending on the intended use of the functionalfragment. For example, a functional fragment of a butyrylcholinesteraseor butyrylcholinesterase variant is intended to refer to a portion ofthe butyrylcholinesterase or butyrylcholinesterase variant that stillretains some or all of the cocaine hydrolysis activity of the parentpolypeptide.

As used herein, the term “library” means a collection of molecules. Alibrary can contain a few or a large number of different molecules,varying from as small as 2 molecules to as large as 10¹³ or moremolecules. Therefore, a library can range in size from 2 to 10, 10 to10², 10² to 10³, 10³ to 10⁵, 10⁵ to 10⁸, 10⁸ to 10¹⁰ or 10¹⁰ to 10¹³molecules. The molecules making up a library can be nucleic acidmolecules such as an RNA, a cDNA or an oligonucleotide; a peptide orpolypeptide including a variant or modified peptide or a peptidecontaining one or more amino acid analogs. In addition, the moleculesmaking up a library can be peptide-like molecules, referred to herein aspeptidomimetics, which mimic the activity of a peptide; or a polypeptidesuch as an enzyme or a fragment thereof. Moreover, a library can bediverse or redundant depending on the intent and needs of the user.Those skilled in the art will know the size and diversity of a librarysuitable for a particular application.

As used herein, the term “region” is intended to refer to an area of theamino acid sequence of butyrylcholinesterase that is determined orpredicted to be important for cocaine hydrolysis activity. As describedbelow, a region has been determined or predicted to be important forcocaine hydrolysis activity by using one or more of structural,biochemical or modeling methods and, as a consequence, is defined bygeneral rather than absolute boundaries. A region can encompass two ormore consecutive amino acid positions of the amino acid sequence ofbutyrylcholinesterase that are predicted to be important for cocainehydrolysis activity. A region of butyrylcholinesterase useful forpracticing the claimed invention is no more than about 30 amino acids inlength and preferably is between 2 and 20, between 5 and 15 amino acidsin length.

As used herein, the term “cocaine hydrolysis activity,” is intended torefer to the catalytic action of a butyrylcholinesterase orbutyrylcholinesterase variant as measured by the rate of cocainehydrolysis into the metabolites.

As used herein, the term “alteration” is intended to refer to amodification at an amino acid position of butyrylcholinesterase. Anamino acid alteration therefore can be a substitution, deletion or anyother structural modification at an amino acid position. An amino acidalteration can occur directly at the amino acid level or result fromtranslation of a nucleic acid encoding an amino acid alteration. Anamino acid alteration can lead to the replacement of an amino acid witha ne*er another amino acid or with an amino acid analog. Examples of anamino acid alteration include the amino acid substitution of Alanine (A)with Tryptophan (W) resulting in the butyryichoinesterase variantdesignated SEQ ID NO: 2; the amino acid substitution of Serine (S) withGlycine (G) resulting in the butyrylcholinesterse variant designated SEQID NO: 4; the amino acid substitution of Proline (P) with Glutamine (Q)resulting in the butyrylcholinesterase variant designated SEQ ID NO: 6;and the amino acid substitution of Proline (P) with Serine (S) resultingin the butyrylcholinesterase variant designated SEQ ID NO: 8.

As used herein, the term “effective amount” is intended to mean anamount of a butyrylcholinesterase variant of the invention that canreduce the cocaine-toxicity or the severity of a cocaine-inducedcondition. Reduction in severity includes, for example, an arrest or adecrease in symptoms, physiological indicators, biochemical markers ormetabolic indicators. Symptoms of cocaine overdose include, for example,cardiac arrythmias, seizures and hypertensive crises. As used herein,the term “treating” is intended to mean causing a reduction in theseverity of a cocaine-induced condition.

As used herein, the term “cocaine-based substrate” refers to (−)-cocaineor any molecule sufficiently similar to (−)-cocaine in structure to behydrolyzed by butyrylcholinesterase or a butyrylcholinesterase variantincluding, for example, (+)-cocaine, acetylcholine, butyrylthiocholine,benzoylcocaine and norcocaine.

The invention provides a butyrylcholinesterase variant comprisingsubstantially the same amino acid sequence shown as SEQ ID NO: 2, orfunctional fragment thereof. The invention also provides abutyrylcholinesterase variant having a 15-fold increase in cocainehydrolysis activity, or functional fragment thereof. The invention alsoprovides a nucleic acid shown as SEQ ID NO: 1, or fragment thereof,which encodes a butyrylcholinesterase variant comprising substantiallythe same amino acid sequence shown as SEQ ID NO: 2.

The invention also provides a butyrylcholinesterase variant comprisingsubstantially the same amino acid sequence shown as SEQ ID NO: 4, orfunctional fragment thereof. The invention also provides abutyrylcholinesterase variant having at least a 4-fold increase incocaine hydrolysis activity, or functional fragment thereof. Theinvention further provides a nucleic acid shown as SEQ ID NO: 3, orfragment thereof, which encodes a butyrylcholinesterase variantcomprising substantially the same amino acid sequence shown as SEQ IDNO: 4. As shown in Table 1, the nucleic acid shown as SEQ ID: 3 differsfrom the nucleic acid encoding human butyrylcholinesterase shown in FIG.3 and designated SEQ ID NO: 16, at positions 1072 through 1074, whichcorrespond to the codon encoding amino acid residue 287. In the humanbutyrylcholinesterase (SEQ ID NO: 16) the codon tca at nucleotidepositions 1072 through 1074 encodes Serine. In contrast, in the nucleicacid encoding the S285G butyrylcholinesterase variant designated SEQ IDNO: 3, the codon ggt at nucleotide positions 1072 through 1074 encodesthe amino acid Glycine.

The invention provides a further butyrylcholinesterase variantcomprising substantially the same amino acid sequence shown as SEQ IDNO: 6, or functional fragment thereof. The invention also provides afurther butyrylcholinesterase variant, having approximately a 4-foldincrease in cocaine hydrolysis activity, or functional fragment thereof.The invention further provides a nucleic acid shown as SEQ ID NO: 5, orfragment thereof, which encodes a butyrylcholinesterase variantcomprising substantially the same amino acid sequence designated SEQ IDNO: 6. As shown in Table 1, the nucleic acid shown as SEQ ID: 5 differsfrom nucleic acid encoding human butyrylcholinesterase shown in FIG. 3and designated SEQ ID NO: 16, at positions 1066 through 1068, whichcorrespond to the codon encoding amino acid residue 285. In the humanbutyrylcholinesterase (SEQ ID NO: 16) the codon cct at nucleotidepositions 1066 through 1068 encodes Proline. In contrast, in the nucleicacid encoding the P285Q butyrylcholinesterase variant designated SEQ IDNO 5, the codon cag at nucleotide positions 1066 through 1068 encodesthe amino acid Glutamine.

The invention provides a further butyrylcholinesterase variantcomprising substantially the same amino acid sequence shown as SEQ IDNO: 8, or functional fragment thereof. The invention also provides afurther butyrylcholinesterase variant, having approximately a three-foldincrease in cocaine hydrolysis activity, or functional fragment thereof.The invention also provides a nucleic acid shown as SEQ ID NO: 7, orfragment thereof, which encodes a butyrylcholinesterase variantcomprising substantially the same amino acid sequence shown as SEQ IDNO: 8. As shown in Table 1, the nucleic acid shown as SEQ ID: 7 differsfrom nucleic acid encoding human butyrylcholinesterase as shown in FIG.3 and designated SEQ ID NO: 16, at positions 1066 through 1068, whichcorrespond to the codon encoding amino acid residue 285. In the humanbutyrylcholinesterase (SEQ ID NO: 16) the codon cct at nucleotidepositions 1066 through 1068 encodes Proline. In contrast, in the nucleicacid encoding P285S butyrylcholinesterase variant designated SEQ ID NO:7, the codon tcg at nucleotide positions 1066 through 1068 encodes theamino acid Serine.

Cholinesterases are ubiquitous, polymorphic carboxylase Type B enzymescapable of hydrolyzing the neurotransmitter acetylcholine and numerousester-containing compounds. Two major cholinesterases areacetylcholinesterase and butyrylcholinesterase. Butyrylcholinesterasecatalyzes the hydrolysis of a number of choline esters as shown:

Butyrylcholinesterase preferentially uses butyrylcholine andbenzoylcholine as substrates. Butyrylcholinesterase is found inmammalian blood plasma, liver, pancreas, intestinal mucosa and the whitematter of the central nervous system. The human gene encodingbutyrylcholinesterase is located on chromosome 3 and over thirtynaturally occuring genetic variations of butyrylcholinesterase areknown. The butyrylcholinesterase polypeptide is 574 amino acids inlength and encoded by 1,722 base pairs of coding sequence. Threenaturally occuring butyrylcholinesterase variations are the atypicalalleles referred to as A variant (SEQ ID NO: 18), the J variant (SEQ IDNO: 19) and the K variant (SEQ ID NO: 20), which are aligned in FIG. 4.The A variant has an D70G mutation and is rare (0.5% allelic frequency),while the J variant has a E497V mutation and has only been found in onefamily. The K variant has a point mutation at nucleotide 1615, whichresults in an A539T mutation and has an allelic frequency of around 12%in Caucasians.

In addition to the naturally-occurring human variations ofbutyrylcholinesterase, a number of species variations are known. Theamino acid sequence of cat butyrylcholinesterase is 88% identical withhuman butyrylcholinesterase (see FIG. 4). Of the seventy amino acidsthat differ, three are located in the active site gorge and are termedA277L, P285L and F398I. Similarly, horse butyrylcholinesterase has threeamino acid differences in the active site compared with humanbutyrylcholinesterase, which are A277V, P285L and F398I (see FIG. 4).The amino acid sequence of rat butyrylcholinesterase contains 6 aminoacid differences in the active site gorge, which are A277K, V280L,T284S, P285I, L286R and V288I (see FIG. 4).

Naturally occurring human butyrylcholinesterase variations, speciesvariations as well as recombinantly prepared mutations have previouslybeen described by Xie et al., Molecular Pharmacology 55:83–91 (1999).Recombinant human butyrylcholinesterase mutants that have been testedfor increased cocaine hydrolysis activity include mutants with thefollowing single or multiple changes: N68Y/Q119/A277W, Q119/V288F/A328Y,Q119Y, E197Q, V288F, A328F, A328Y, F329A and F329S. Out of thesemutants, the only butyrylcholinesterase mutant identified that exhibitsincreased cocaine hydrolysis activity is the A328Y mutant, which has aTyrosine (Y) rather than an Alanine (A) at amino acid position 328 andexhibits a four-fold increase in cocaine hydrolysis activity compared tohuman butyrylcholinesterase (Xie et al., supra, 1999).

The invention provides a butyrylcholinesterase variant shown as SEQ IDNO: 2 that has substantially the same amino acid sequence as humanbutyrylcholinesterase, but includes at amino acid position 328 of humanbutyrylcholinesterase (SEQ ID NO: 17) a Tryptophan (W) substitution inplace of the encoded Alanine (A) residue. The A328Wbutyrylcholinesterase variant (SEQ ID NO: 2) was obtained by PCRsite-directed mutagenesis of human butyrylcholinesterase as described inExample I below and exhibits at least a fifteen-fold increase in cocainehydrolysis activity compared to human butyrylcholinesterase.

The invention further provides a butyrylcholinesterase variant shown asSEQ ID NO: 4 that has substantially the same amino acid sequence ashuman butyrylcholinesterase, but includes at amino acid position 287 ofhuman butyrylcholinesterase (SEQ ID NO: 17) a Glycine (G) substitutionin place of the Serine (S) residue encoded in humanbutyrylcholinesterase. The S287G butyrylcholinesterase variant (SEQ IDNO: 4) is encoded by a nucleotide sequence (SEQ ID NO: 3) that issubstantially the same as that of human butyrylcholinesterase (SEQ IDNO:16), but has the codon ggt encoding the amino acid Glycine instead ofthe codon tca encoding Serine at the nucleotide positions correspondingto position 287 of human butyrylcholinesterase (SEQ ID NO: 17). TheS287G butyrylcholinesterase variant (SEQ ID NO: 4) was obtained asdescribed in Examples II through VI below and exhibits at least afour-fold increase in cocaine hydrolysis activity compared to humanbutyrylcholinesterase.

The invention provides a butyrylcholinesterase variant shown as SEQ IDNO: 6 that has substantially the same amino acid sequence as humanbutyrylcholinesterase, but includes at amino acid position 285 of humanbutyrylcholinesterase (SEQ ID NO: 17) a Glutamine (Q) substitution inplace of the encoded Proline (P) residue. The P285Qbutyrylcholinesterase variant (SEQ ID NO: 6) is encoded by a nucleotidesequence (SEQ ID NO: 5) that is substantially the same as that of humanbutyrylcholinesterase (SEQ ID NO: 16), but has the codon cag encodingthe amino acid Glutamine instead of the codon cct encoding Proline atthe nucleotide positions corresponding to position 285 of humanbutyrylcholinesterase (SEQ ID NO: 17). The P285Q butyrylcholinesterasevariant (SEQ ID NO: 6) was obtained as described in Examples II throughVI below and exhibits an approximately four-fold increase in cocainehydrolysis activity compared to human butyrylcholinesterase.

The invention also provides a butyrylcholinesterase variant shown as SEQID NO: 8 that has substantially the same amino acid sequence as humanbutyrylcholinesterase, but includes at amino acid position 285 of humanbutyrylcholinesterase (SEQ ID NO: 16) a Serine (S) substitution in placeof the encoded Proline (P) residue. The P285S butyrylcholinesterasevariant (SEQ ID NO: 8) is encoded by a nucleotide sequence (SEQ ID NO:7) that is substantially the same as that of human butyrylcholinesterase(SEQ ID NO: 16), but has the codon tcg encoding the amino acid Serineinstead of the codon cct encoding Proline at the nucleotide positionscorresponding to position 287 of human butyrylcholinesterase (SEQ ID NO:17). The P285S butyrylcholinesterase variant (SEQ ID NO: 8) was obtainedas described in Examples II through VI below and exhibits anapproximately three-fold increase in cocaine hydrolysis activitycompared to human butyrylcholinesterase.

TABLE 1 Nucleotide Sequences Corresponding to Amino Acid 284 throughamino acid 288. Human BchE (SEQ ID NO:25) act cct ttg tca gta S287G (SEQID NO:26) act cct ttg ggt gta P285Q (SEQ ID NO:27) act cag ttg tca gtaP285S (SEQ ID NO:28) act tcg ttg tca gta

A butyrylcholinesterase variant of the invention can be prepared by avariety of methods well known in the art. If desired, random mutagenesiscan be performed to prepare a butyrylcholinesterase variant of theinvention. Alternatively, as disclosed herein, site directed mutagenesisbased on the information obtained from structural, biochemical andmodeling methods described herein can be performed to target those aminoacids predicted to be important for cocaine hydrolysis activity. Forexample, molecular modeling of cocaine in the active site ofbutyrylcholinesterase can be utilized to predict amino acid alterationsthat allow for higher catalytic efficiency based on a better fit betweenthe enzyme and its substrate. As described herein, residues predicted tobe important for cocaine hydrolysis activity Include 8 hydrophobic gorgeresidues and the catalytic triad residues. Furthermore, it is understoodthat amino acid alterations of residues important for the functionalstructure of a butyrylcholinesterese variant which include the cysteineresidues ⁶⁵Cys-⁹²Cys, ²⁵²Cys-²⁶³Cys, and ⁴⁰⁰Cys-⁵¹⁹Cys involved inintrachain disulfide bonds are generally not altered in the preparationof a butyrylcholinesterase variant that has cocaine hydrolysis activity.

Following mutagenesis of butyrylcholinesterase or abutyrylcholinesterase variant expression, purification and functionalcharacterization of the butyrylcholinesterase variant can be performedby methods well known in the art. As disclosed below, abutyrylcholinesterase variant can be expressed in an appropriate hostcell line and subsequently purified and characterized for cocainehydrolysis activity. Butyrylcholinesterase variants characterized ashaving significantly increased cocaine hydrolysis activity cansubsequently be used in the methods of hydrolyzing a cocaine-basedsubstrate as well as the methods of treating a cocaine-induced conditiondescribed below.

A butyrylcholinesterase variant of the invention exhibits cocainehydrolysis activity. As disclosed herein, a butyrylcholinesterasevariant of the invention can have enhanced cocaine hydrolysis activityand can be used to treat a cocaine-induced condition. A polypeptidehaving minor modifications compared to a butyrylcholinesterase variantof the invention is encompassed by the invention so long as equivalentcocaine hydrolysis activity is retained. In addition, functionalfragments of a butyrylcholinesterase variant that still retain some orall of the cocaine hydrolysis activity of the parentbutyrylcholinesterase variant are similarly included in the invention.Similarly, functional fragments of nucleic acids, which encodefunctional fragments of a butyrylcholinesterase variant of the inventionare similarly encompassed by the invention.

A functional fragment of a butyrylcholinesterase or abutyrylcholinesterase variant of the invention can be prepared byrecombinant methods involving expression of a nucleic acid moleculeencoding the butyrylcholinesterase variant or functional fragmentthereof, followed by isolation of the variant or functional fragmentthereof by routine biochemical methods described herein. It isunderstood that functional fragments can also be prepared by enzymaticor chemical cleavage of the full length butyrylcholinesterase variant.Methods for enzymatic and chemical cleavage and for purification of theresultant peptide fragments are well known in the art (see, for example,Deutscher, Methods in Enzymology, Vol. 182, “Guide to ProteinPurification,” San Diego: Academic Press, Inc. (1990), which isincorporated herein by reference).

Furthermore, functional fragments of a butyrylcholinesterase variant canbe produced by chemical synthesis. If desired, such molecules can bemodified to include D-stereoisomers, non-naturally occurring aminoacids, and amino acid analogs and mimetics in order to optimize theirfunctional activity, stability or bioavailability. Examples of modifiedamino acids and their uses are presented in Sawyer, Peptide Based DrugDesign, ACS, Washington (1995) and Gross and Meienhofer, The Peptides:Analysis, Synthesis, Biology, Academic Press, Inc., New York (1983),both of which are incorporated herein by reference.

If desired, random segments of a butyrylcholinesterase variant can beprepared and tested in the assays described herein. A fragment havingany desired boundaries and modifications compared to the amino acidsequence of the reference butyrylcholinesterase or butyryloholinesterasevariant of the invention can be prepared. Alternatively, availableinformation obtained by the structural, biochemical and modeling methodsdescribed herein can be used to prepare only those fragments of abutyrylcholinesterase variant that are likely to retain the cocainehydrolysis activity of the parent variant. As described herein, residuespredicted to be important for cocaine hydrolysis activity include 8hydrophobic gorge residues and the catalytic triad residues.Furthermore, residues important for the functional structure of abutyrylcholinesterase variant include the cysteine residues ⁶⁵Cys-⁹²Cys,²⁵²Cys- ²⁶³Cys, and ⁴⁰⁰Cys-⁵¹⁹ Cys involved in intrachain disulfidebonds. Therefore, a functional fragment can be a truncated form, regionor segment of the reference butyrylcholinesterase variant designed topossess most or all of the residues critical for cocaine hydrolysisactivity or functional structure so as to retain equivalent cocainehydrolysis activity. Similarly, a functional fragment can includenon-peptidic structural elements that serve to mimic structurally orfunctionally important residues of the reference variant. Also includedas butyrylcholinesterase variants of the invention are fusion proteinsthat result from linking a butyrylcholinesterase variant or functionalfragment thereof to a heterologous protein, such as a therapeuticprotein, as well as fusion constructs of nucleic acids encoding suchfusion proteins. Fragments of nucleic acids that can hybridize to abutyrylcholinesterase variant or functional fragment thereof are useful,for example, as hybridization probes and are also encompassed by theclaimed invention.

Thus, the invention provides four butyrylcholinesterase variantscomprising substantially the same amino acid sequences shown as SEQ IDNOS: 2, 4, 6, and 8, respectively, or functional fragment thereof. Theinvention also provides a butyrylcholinesterase variant having a 15-foldincrease in cocaine hydrolysis activity, or functional fragment thereof;a butyrylcholinesterase variant having at least a four-fold increase incocaine hydrolysis activity, or functional fragment thereof; abutyrylcholinesterase variant having approximately 4-fold increase incocaine hydrolysis activity, or functional fragment thereof; and abutyrylcholinesterase variant having approximately a three-fold increasein cocaine hydrolysis activity, or functional fragment thereof. Theinvention also provides four nucleic acids shown as SEQ ID NO: 1, 3, 5,and 7, respectively, or fragment thereof, which encode thebutyrylcholinesterase variants comprising substantially the same aminoacid sequences shown as SEQ ID NO: 2, 4, 6, and 8, respectively.

The invention also provides a library of butyrylcholinesterase variantshaving at least one amino acid alteration in one or more regions ofbutyrylcholinesterase corresponding to amino acid positions 68–82 (SEQID NO: 9), 110–121 (SEQ ID NO: 10), 194–201 (SEQ ID NO: 11), 224–234(SEQ ID NO: 12), 277–289 (SEQ ID NO: 13), 327–332 (SEQ ID NO: 14) or429–442 (SEQ ID NO: 15) of butyrylcholinesterase or functional fragmenttherof, wherein the library of butyrylcholinesterase variants of theinvention has at least one butyrylcholinesterase variant exhibitingenhanced cocaine hydrolysis activity compared to butyrylcholinesterase,with the proviso that a butyrylcholinesterase variant having a singleamino acid alteration is not the human butyrylcholinesterase having Y atposition 328. The invention further provides a library ofbutyrylcholinesterase variants wherein said butyrylcholinesterasevariants have at least two amino acid alterations.

In addition, the invention provides seven distinct libraries ofbutyrylcholinesterase variants, each variant having at least one aminoacid alteration in a region of butyrylcholinesterase corresponding toamino acid positions 68–82 (SEQ ID NO: 9), 110–121 (SEQ ID NO: 10),194–201 (SEQ ID NO: 11), 224–234 (SEQ ID NO: 12), 277–289 (SEQ ID NO:13), 327–332 (SEQ ID NO: 14) or 429–442 (SEQ ID NO: 15) ofbutyrylcholinesterase or functional fragment thereof, respectively. Alibrary of butyrylcholinesterase variants of the invention can be usedto screen for butyrylcholinesterase variants with increased cocainehydrolysis activity.

A library that is sufficiently diverse to contain abutyrylcholinesterase variant with enhanced cocaine hydrolysis activitycan be prepared by a variety of methods well known in the art. Thoseskilled in the art will know what size and diversity is necessary orsufficient for the intended purpose. For example, a library ofbutyrylcholinesterase variants can be prepared that contains each of the19 amino acids not found in the reference butyrylcholinesterase at eachof the approximately 573 amino acid positions and screening theresultant variant library for butyrylcholinesterase variants withenhanced cocaine hydrolysis activity.

Alternatively, a focused library can be prepared utilizing thestructural, biochemical and modeling information relating tobutyrylcholinesterase as described herein. It is understood that anyinformation relevant to the determination or prediction of residues orregions important for the cocaine hydrolysis activity or structuralfunction of butyrylcholinesterase can be useful in the design of afocused library of butyrylcholinesterase variants of the invention.Thus, the butyrylcholinesterase variants that make up the library ofbutyrylcholinesterase variants of the invention can contain amino acidalterations at amino acid positions located in regions determined orpredicted to be important for cocaine hydrolysis activity. A focusedlibrary of butyrylcholinesterase variants is desirable as itsignificantly decreases the number of variants that need to be screenedin order to identify a butyrylcholinesterase variant with enhancedcocaine hydrolysis activity by targeting amino acid alterations toregions determined or predicted to be important for cocaine hydrolysisactivity.

Regions important for the cocaine hydrolysis activity ofbutyrylcholinesterase can be determined or predicted through a varietyof methods known in the art and used to focus the synthesis of a libraryof butyrylcholinesterase variants. Related enzymes such as, for example,acetylcholinesterase and carboxylesterase, that share a high degree ofsequence similarity and have biochemically similar catalytic propertiescan provide information regarding the regions important for catalyticactivity of butyrylcholinesterase. For example, structural modeling canreveal the active site of an enzyme, which is a three-dimensionalstructure such as a cleft, gorge or crevice formed by amino acidresidues generally located apart from each other in primary structure.Therefore, amino acid residues that make up regions ofbutyrylcholinesterase important for cocaine hydrolysis activity caninclude residues located along the active site gorge. For a descriptionof structural modeling of butyrylcholinesterase, see for example, Harelet al., Proc. Nat. Acad. Sci. USA 89: 10827–10831 (1992) and Soreq etal., Trends Biochem. Sci. 17(9): 353–358 (1992), which are incorporatedherein by reference.

In addition to structural modeling of butyrylcholinesterase, biochemicaldata can be used to determine or predict regions ofbutyrylcholinesterase important for cocaine hydrolysis activity whenpreparing a focused library of butyrylcholinesterase variants. In thisregard, the characterization of naturally occurringbutyrylcholinesterase variants with altered cocaine hydrolysis activityis useful for identifying regions important for the catalytic activityof butyrylcholinesterase. Similarly, site-directed mutagenesis studiescan provide data regarding catalytically important amino acid residuesas reviewed, for example, in Schwartz et al; Pharmac. Ther. 67: 283–322(1992), which is incorporated by reference.

To generate a library of butyrylcholinesterase variants of the inventiondistinct types of information can be used alone or combined to determineor predict a region of an amino acid sequence of butyrylcholinesteraseimportant for cocaine hydrolysis activity. For example, informationbased on structural modeling and biochemical data is combined todetermine a region of an amino acid sequence of butyrylcholinesteraseimportant for cocaine hydrolysis activity. Because information obtainedby a variety of methods can be combined to predict the catalyticallyactive regions, one skilled in the art will appreciate that the regionsthemselves represent approximations rather than strict confines. As aresult, a library of butyrylcholinesterases can containbutyrylcholinesterase variants that have amino acid alterations outsideof the regions determined or predicted to be important for cocainehydrolysis activity. Similarly, a butyrylcholinesterase variant of theinvention can have amino acid alterations outside of the regionsdetermined or predicted to be important for cocaine hydrolysis activity.Furthermore, a butyrylcholinesterase variant of the invention can haveany other modification that does not significantly change its cocainehydrolysis activity. It is further understood that the number of regionsdetermined or predicted to be important for cocaine hydrolysis activitycan vary based on the predictive methods used.

Once a number of regions has been identified by any method appropriatefor determination of regions important for cocaine hydrolysis, orcombination thereof, each region can be randomized across some or allamino acid positions to create a library of variants containing thewild-type amino acid plus one or more of the other nineteen naturallyoccurring amino acids at one or more positions within each of theregions. Seven regions of an amino acid sequence ofbutyrylcholinesterase selected for the focused library ofbutyrylcholinesterase variants provided by the invention are shown inTable 2.

TABLE 2 Summary of Butyrylcholinesterase Libraries Region LocationLength # Variants Species Diversity 1 68–82 15 285 3 2 110–121 12 228 33 194–201 8 152 1 4 224–234 11 209 2 5 277–289 13 247 8 6 327–332 6 1140 7 429–442 14 266 0 Total 79 1,501 13.8%

The location of the regions of the amino acid sequence ofbutyrylcholinesterase shown in Table 2 are shown in reference to theamino acid sequence of human butyrylcholinesterase (FIG. 2). The numberof butyrylcholinesterase variants for each region reflects one variantfor each of 19 amino acid substitutions at each position compared tohuman butyrylcholinesterase and a total library size of 1,501 variants.Species diversity indicates the number of positions within each regionthat have a naturally occurring amino acid difference compared to humanbutyrylcholinesterase.

Methods for preparing libraries containing diverse populations ofvarious types of molecules such as peptides, peptoids andpeptidoinimetics are well known in the art (see, for example, Ecker andCrooke, Biotechnology 13:351–360(1995), and Blondelle et al., TrendsAnal. Chem. 14:83–92 (1995), and the references cited therein, each ofwhich is incorporated herein by reference; see, also, Goodman and Ro,Peptidomimetics for Drug Design, in “Burger's Medicinal Chemistry andDrug Discovery” Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages803–861, and Gordon et al., J. Med. Chem. 37:1385–1401 (1994), each ofwhich is incorporated herein by reference). Where a molecule is apeptide, protein or fragment thereof, the molecule can be produced invitro directly or can be expressed from a nucleic acid, which can beproduced in vitro. Methods of synthetic peptide chemistry are well knownin the art.

A library of butyrylcholinesterase variants can be produced, forexample, by constructing a nucleic acid expression library encodingbutyrylcholinesterase variants. Methods for producing such libraries arewell known in the art (see, for example, Sambrook et al., MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989),which is incorporated herein by reference). A library of nucleic acidsencoding butyrylcholinesterase variants can be composed of DNA, RNA oranalogs thereof. A library containing RNA molecules can be constructed,for example, by synthesizing the RNA molecules chemically.

The invention further provides seven distinct libraries of nucleic acidsencoding butyrylcholinesterase variants, each nucleic acid having atleast one codon encoding at least one amino acid alteration in a regionof butyrylcholinesterase corresponding to amino acid positions 68–82(SEQ ID NO: 9), 110–121 (SEQ ID NO: 10), 194–201 (SEQ ID NO: 11),224–234 (SEQ ID NO: 12), 277–289 (SEQ ID NO: 13), 327–332 (SEQ ID NO:14) or 429–442 (SEQ ID NO: 15) of butyrylcholinesterase, respectively.

The generation of a library of nucleic acids encodingbutyrylcholinesterase variants can be by any means desired by the user.Those skilled in the art will know what methods can be used to generatelibraries of nucleic acids encoding butyrylcholinesterase variants. Forexample, butyrylcholinesterase variants can be generated by mutagenesisof nucleic acids encoding butyrylcholinesterase using methods well knownto those skilled in the art (Molecular Cloning: A Laboratory Manual,Sambrook et al; eds; Cold Spring Harbor Press, Plainview, N.Y. (1989)).A library of nucleic acids encoding butyrylcholinesterase variants ofthe invention can be randomized to be sufficiently diverse to containnucleic acids encoding every possible naturally occurring amino acid ateach amino acid position of butyrylcholinesterase. Alternatively, alibrary of nucleic acids can be prepared such tat it contains nucleicacids encoding every possible naturally occurring amino acid at eachamino acid only at positions located within a region ofbutyrylcholinesterase predicted or determined to be important forcocaine hydrolysis activity.

One or more mutations can be introduced into a nucleic acid moleculeencoding a butyrylcholinesterase variant to yield a modified nucleicacid molecule using, for example, site-directed mutagenesis (see Wu(Ed.), Meth. In Enzymol. Vol. 217, San Diego: Academic Press (1993);Higuchi, “Recombinant PCR” in Innis et al. (Ed.), PCR Protocols, SanDiego: Academic Press, Inc. (1990), each of which is incorporated hereinby reference). Such mutagenesis can be used to introduce a specific,desired amino acid alteration. Thus, distinct libraries containing aminoacid alterations in one or more of the regions determined to beimportant for cocaine hydrolysis activity as well as a single librarycontaining mutations in several or all of the regions can be prepared.

The efficient synthesis and expression of libraries ofbutyrylcholinesterase variants using oligonucleotide-directedmutagenesis can be accomplished as previously described by Wu et al.,Proc. Natl. Acad. Sci. USA, 95:6037–6042 (1998); Wu et al., J. Mol.Biol., 294:151–162 (1999); and Kunkel, Proc. Natl. Acad. Sci. USA,82:488–492 (1985), which are incorporated herein by reference.Oligonucleotide-directed mutagenesis is a well-known and efficientprocedure for systematically introducing mutations, independent of theirphenotype and is, therefore, ideally suited for directed evolutionapproaches to protein engineering. To perform oligonucleotide-directedmutagenesis a library of nucleic acids encoding the desired mutations ishybridized to single-stranded uracil-containing template of thewild-type sequence. The methodology is flexible, permitting precisemutations to be introduced without the use of restriction enzymes, andis relatively inexpensive if oligonucleotides are synthesized usingcodon-based mutagenesis.

Codon-based synthesis or mutagenesis represents one method well known inthe art for avoiding genetic redundancy while rapidly and efficientlyproducing a large number of alterations in a known amino acid sequenceor for generating a diverse population of random sequences. This methodis the subject matter of U.S. Pat. Nos. 5,264,563 and 5,523,388 and isalso described in Glaser et al. J. Immunology 149:3903–3913 (1992).Briefly, coupling reactions for the randomization of, for example, alltwenty codons which specify the amino acids of the genetic code areperformed in separate reaction vessels and randomization for aparticular codon position occurs by mixing the products of each of thereaction vessels. Following mixing, the randomized reaction productscorresponding to codons encoding an equal mixture of all twenty aminoacids are then divided into separate reaction vessels for the synthesisof each randomized codon at the next position. If desired, equalfrequencies of all twenty amino acids can be achieved with twentyvessels that contain equal portions of the twenty codons. Thus, it ispossible to utilize this method to generate random libraries of theentire sequence of butyrylcholinesterase or focused libraries of theregions determined or predicted to be important for cocaine hydrolysisactivity.

Variations to the above synthesis method also exist and include, forexample, the synthesis of predetermined codons at desired positions andthe biased synthesis of a predetermined sequence at one or more codonpositions as described by Wu et al, supra, 1998. Biased synthesisinvolves the use of two reaction vessels where the predetermined orparent codon is synthesized in one vessel and the random codon sequenceis synthesized in the second vessel. The second vessel can be dividedinto multiple reaction vessels such as that described above for thesynthesis of codons specifying totally random amino acids at aparticular position. Alternatively, a population of degenerate codonscan be synthesized in the second reaction vessel such as through thecoupling of NNG/T nucleotides or NNX/X where N is a mixture of all fournucleotides. Following synthesis of the predetermined and random codons,the reaction products in each of the two reaction vessels are mixed andthen redivided into an additional two vessels for synthesis at the nextcodon position.

A modification to the above-described codon-based synthesis forproducing a diverse number of variant sequences can similarly beemployed for the production of the libraries of butyrylcholinesterasevariants described herein. This modification is based on the two vesselmethod described above which biases synthesis toward the parent sequenceand allows the user to separate the variants into populations containinga specified number of codon positions that have random codon changes.

Briefly, this synthesis is performed by continuing to divide thereaction vessels after the synthesis of each codon position into two newvessels. After the division, the reaction products from each consecutivepair of reaction vessels, starting with the second vessel, is mixed.This mixing brings together the reaction products having the same numberof codon positions with random changes. Synthesis proceeds by thendividing the products of the first and last vessel and the newly mixedproducts from each consecutive pair of reaction vessels and redividinginto two new vessels. In one of the new vessels, the parent codon issynthesized and in the second vessel, the random codon is synthesized.For example, synthesis at the first codon position entails synthesis ofthe parent codon in one reaction vessel and synthesis of a random codonin the second reaction vessel. For synthesis at the second codonposition, each of the first two reaction vessels is divided into twovessels yielding two pairs of vessels. For each pair, a parent codon issynthesized in one of the vessels and a random codon is synthesized inthe second vessel. When arranged linearly, the reaction products in thesecond and third vessels are mixed to bring together those productshaving random codon sequences at single codon positions. This mixingalso reduces the product populations to three, which are the startingpopulations for the next round of synthesis. Similarly, for the third,fourth and each remaining position, each reaction product population forthe preceding position are divided and a parent and random codonsynthesized.

Following the above modification of codon-based synthesis, populationscontaining random codon changes at one, two, three and four positions aswell as others can be conveniently separated out and used based on theneed of the individual. Moreover, this synthesis scheme also allowsenrichment of the populations for the randomized sequences over theparent sequence since the vessel containing only the parent sequencesynthesis is similarly separated out from the random codon synthesis.This method can be used to synthesize a library of nucleic acidsencoding butyrylcholinesterase variants having amino acid alterations inone or more regions of butyrylcholinesterase predicted to be importantfor cocaine hydrolysis activity.

Alternatively, a library of nucleic acids encoding butyrylcholinesterasevariants can also be generated using gene shuffling. Gene shuffling orDNA shuffling is a method for directed evolution that generatesdiversity by recombination (see, for example, Stemmer, Proc. Natl. Acad.Sci. USA 91:10747–10751 (1994); Stemmer, Nature 370:389–391 (1994);Crameri et al., Nature 391:288–291 (1998); Stemmer et al., U.S. Pat. No.5,830,721, issued Nov. 3, 1998). Gene shuffling or DNA shuffling is amethod using in vitro homologous recombination of pools of selectedmutant genes. For example, a pool of point mutants of a particular genecan be used. The genes are randomly fragmented, for example, usingDNase, and reassembled by PCR. If desired, DNA shuffling can be carriedout using homologous genes from different organisms to generatediversity (Crameri et al., supra, 1998). The fragmentation andreassembly can be carried out in multiple rounds, if desired. Theresulting reassembled genes constitute a library ofbutyrylcholinesterase variants that can be used in the inventioncompositions and methods.

Thus, the invention also provides a library of nucleic acids encodingbutyrylcholinesterase variants, each nucleic acid having at least onecodon encoding at least one amino acid alteration in one or more regionsof butyrylcholinesterase corresponding to amino acid positions 68–82(SEQ ID NO: 9), 110–121 (SEQ ID NO: 10), 194–201 (SEQ ID NO: 11),224–234 (SEQ ID NO: 12), 277–289 (SEQ ID NO: 13), 327–332 (SEQ ID NO:14) or 429–442 (SEQ ID NO: 15) of butyrylcholinesterase, wherein atleast one of the nucleic acids encodes a butyrylcholinesterase varianthaving enhanced cocaine hydrolysis activity compared tobutyrylcholinesterase, with the proviso that a butyrylcholinesterasevariant having a single amino acid alteration is not the humanbutyrylcholinesterase having Y at position 328.

The invention library of nucleic acids encoding butyrylcholinesterasevariants can be expressed in a variety of eukaryotic cells. For example,the nucleic acids can be expressed in mammalian cells, insect cells,plant cells, and non-yeast fungal cells. Mammalian cell lines useful forexpressing the invention library of nucleic acids encodingbutyrylcholinesterase variants include, for example, Chinese HamsterOvary (CHO), human T293 and Human NIH 3T3 cell lines. Expression of theinvention library of nucleic acids encoding butyrylcholinesterasevariants can be achieved by both stable or transient cell transfection(see Example III, Table 5).

The incorporation of variant nucleic acids or heterologous nucleic acidfragments at an identical site in the genome functions to createisogenic cell lines that differ only in the expression of a particularvariant or heterologous nucleic acid. Incorporation at a single siteminimizes positional effects from integration at multiple sites in agenome that affect transcription of the mRNA encoded by the nucleic acidand complications from the incorporation of multiple copies orexpression of more than one nucleic acid species per cell. Techniquesknown in the art that can be used to target a variant or a heterologousnucleic acid to a specific location in the genome include, for example,homologous recombination, retroviral targeting and recombinase-mediatedtargeting.

One approach for targeting variant or heterologous nucleic acids to asingle site in the genome uses Cre recombinase to target insertion ofexogenous DNA into the eukaryotic genome at a site containing a sitespecific recombination sequence (Sauer and Henderson, Proc. Natl. Acad.Sci. USA, 85:5166–5170 (1988); Fukushige and Sauer, Proc. Natl. Acad.Sci. U.S.A. 89:7905–7909 (1992); Bethke and Sauer, Nuc. Acids Res.,25:2828–2834 (1997)). In addition to Cre recornbinase, Flp recombinasecan also be used to target insertion of exogenous DNA into a particularsite in the genome (Dymecki, Proc. Nati. Acad. Sci. U.S.A. 93:6191–6196(1996)). The target site for Fip recombinase consists of 13 base-pairrepeats separated by an 8 base-pair spacer: 5′-GAAGTTCCTATTC[TCTAGAAA]GTATAGGAACTTC-3′ (SEQ ID NO: 24). As described herein, thebutyrylcholinesterases designated SEQ ID NOS: 4, 6, and 8, were obtainedby transfection of variant libraries corresponding to region 5 of humanbutyrylcholinesterase (see, Table 2) into mammalian cells using Flprecornbinase and the human 293T cell line. It is understood that anycombination of site-specific recombinase and corresponding recombinationsite can be used in methods of the invention to target a nucleic acid toa particular site in the genome.

A suitable recombinase can be encoded on a vector that is co-transfectedwith a vector containing a nucleic acid encoding a butyrylcholinesterasevariant. Alternatively, the expression element of a recombinase can beincorporated into the same vector expressing a nucleic acid encoding abutyrylcholinesterase variant. In addition to simultaneouslytransfecting the nucleic acid encoding a recombinase with the nucleicacids encoding a butyrylcholinesterase variant, a vector encoding therecombinase can be transfected into a cell, and the cells can beselected for expression of recombinase. A cell stably expressing therecombinase can subsequently be transfected with nucleic acids encodingvariant nucleic acids.

As disclosed herein, the precise site-specific DNA recombinationmediated by Cre recombinase can be used to create stable mammaliantransformants containing a single copy of exogenous DNA encoding abutyrylcholinesterase variant. As exemplified below, the frequency ofCre-mediated targeting events can be enhanced substantially using amodified doublelox strategy. The doublelox strategy is based on theobservation that certain nucleotide changes within the core region ofthe lox site alter the site selection specificity of Cre-mediatedrecombination with little effect on the efficiency of recombination(Hoess et al., Nucleic Acids Res. 14:2287–2300 (1986)). Incorporation ofloxP and an altered loxP site, termed lox511, in both the targetingvector and the host cell genome results in site-specific recombinationby a double crossover event. The doublelox approach increases therecovery of site-specific integrants by 20-fold over the singlecrossover insertional recombination, increasing the absolute frequencyof site-specific recombination such that it exceeds the frequency ofillegitimate recombination (Bethke and Sauer, Nuc. Acids Res.,25:2828–2834 (1997)).

Following the expression of a library of butyrylcholinesterase variantsin a mammalian cell line, randomly selected clones can be sequenced andscreened for increased cocaine hydrolysis activity. Methods forsequencing selected clones are well known to those of skill in the artand are described, for example, in Sambrook et al., supra, 1992, and inAusubel et al; supra, 1998. Selecting a suitable method for measuringthe cocaine hydrolysis activity of a butyrylcholinesterase variantdepends on a variety of factors such as, for example, the amount of thebutyrylcholinesterase variant that is available. The cocaine hydrolysisactivity of a butyrylcholinesterase variant can be measured, forexample, by spectrophotometry, by a microtiter-based assay utilizing apolyclonal anti-butyrylcholinesterase antibody to uniformly capture thebutryrylcholinesterase variants and by high-performance liquidchromatography (HPLC).

Enhanced cocaine hydrolysis activity of a butyrylcholinesterase variantcompared to butyrylcholinesterase can be determined by a comparison ofcatalytic efficiencies as described in Example I. Clones expressingbutyrylcholinesterase variants exhibiting increased cocaine hydrolysisactivity are sequenced to reveal the precise location and nature of themutation. To ensure that a library of butyrylcholinesterase variants hasbeen screened exhaustively, screening of each library can be continueduntil clones encoding identical butyrylcholinesterase amino acidalterations have been identified on multiple occasions.

Clones expressing a butyrylcholinesterase variant with increased cocainehydrolysis activity can be used to establish larger-scale culturessuitable for purifying larger quantities of the butyrylcholinesterase. Abutyrylcholinesterase variant of interest can be cloned into anexpression vector and used to transfect a cell line, which cansubsequently be expanded. Those skilled in the art will know what typeof expression vector is suitable for a particular application. Abutyrylcholinesterase variant exhibiting increased cocaine hydrolysisactivity can be cloned, for example, into an expression vector carryinga gene that confers resistance to a particular chemical agent to allowpositive selection of the transfected cells. An expression vectorsuitable for transfection of, for example, mammalian cell lines cancontain a promoter such as the cytomegaloviras (CMV) promoter forselection in mammalian cells. As described herein, abutyrylcholinesterase variant can be cloned into a mammalian expressionvector and transfected into Chinese Hamster Ovary cells (CHO).Expression vectors suitable for expressing a butyrylcholinesterasevariant are well known in the art and commercially available.

Clones expressing butyrylcholinesterase variants can be selected andtested for cocaine hydrolysis activity. Cells carrying clones exhibitingenhanced cocaine hydrolysis activity can be expanded by routine cellculture systems to produce larger quantities of a butyrylcholinesterasevariant of interest. The concentrated recombinant butyrylcholinesterasevariant can be harvested and purified by methods well known in the artand described, for example, by Masson et al., Biochemistry 36: 2266–2277(1997), which is incorporated herein by reference.

A butyrylcholinesterase variant exhibiting increased cocaine hydrolysisactivity in vitro can be utilized for the treatment of cocaine toxicityand addiction in vivo. The potency for treating cocaine toxicity of abutyrylcholinesterase variant exhibiting increased cocaine hydrolysisactivity in vitro can be tested using an acute overdose animal model asdisclosed herein (see Example VII). In addition, animal models ofreinforcement and discrimination are used to predict the efficacy of abutyrylcholinesterase variant for treatment of cocaine addiction asdisclosed below (see Example VII). Suitable animal subjects for overdoseas well as reinforcement and discrimination models are known in the artand include, for example, rodent and primate models. Abutyrylcholinesterase variant effective in reducing either cocainetoxicity or cocaine addiction in one or more animal models can be usedto treat a cocaine-induced condition by administering an effectiveamount of the butyrylcholinesterase variant to an individual.

A butyrylcholinesterase variant having an increased serum half-life canbe useful for testing a butyrylcholinesterase variant in a subject ortreating a cocaine-induced condition in an individual. Useful methodsfor increasing the serum half-life of a butyrylcholinesterase variantinclude, for example, conversion of the butyrylcholinesterase variantinto a tetramer, covalently attaching synthetic and natural polymerssuch as polyethylene glycol (PEG) and dextrans to the truncatedbutyrylcholinesterase variant, liposome formulations, or expression ofthe enzyme as an Ig-fusion protein. As disclosed herein, conversion of abutyrylcholineserase variant into a tetramer can be achieved byco-transfecting the host cell line with the COLQ gene (Example I) aswell as by addition of poly-L-proline to the media of transfected cells.These and other methods known in the art for increasing the serumhalf-life of a butyrylcholinesterase variant are useful for testing abutyrylcholinesterase variant in an animal subject or treating acocaine-induced condition in an individual.

The invention also provides a method of hydrolyzing a cocaine-basedbutyrylcholinesterase substrate comprising contacting abutyrylcholinesterase substrate with the butyrylcholinesterase variantshown as SEQ ID NO: 2 under conditions that allow hydrolysis of cocaineinto metabolites, wherein the butyrylcholinesterase variant exhibits afive-fold or more increase in cocaine hydrolysis activity compared tobutyrylcholinesterase. In addition, the invention provides a method oftreating a cocaine-induced condition comprising administering to anindividual an effective amount of a butyrylcholinesterase variant (SEQID NO: 2) exhibiting increased cocaine hydrolysis activity compared tobutyrylcholinesterase.

The invention further provides a method of hydrolyzing a cocaine-basedbutyrylcholinesterase substrate comprising contacting abutyrylcholinesterase substrate with a butyrylcholinesterase variantselected from the group shown as SEQ ID NO: 4, SEQ ID NO: 6 and SEQ IDNO: 8, under conditions that allow hydrolysis of cocaine intometabolites, wherein the butyrylcholinesterase variant exhibits atwo-fold or more increase in cocaine hydrolysis activity compared tobutyrylcholinesterase. In addition, the invention provides a method oftreating a cocaine-induced condition comprising administering to anindividual an effective amount of a butyrylcholinesterase variantselected from the group shown as SEQ ID NO: 4, SEQ ID NO: 6 and SEQ IDNO: 8, exhibiting increased cocaine hydrolysis activity compared tobutyrylcholinesterase.

As described herein, a butyrylcholinesterase variant exhibitingincreased cocaine hydrolysis activity can hydrolyze a cocaine-basedbutyrylcholinesterase substrate in vitro as well as in vivo. Acocaine-based butyrylcbolinesterase substrate can be contacted wit abntyrylcholinesterase variant of the invention in vitro, for example, byadding the substrate to supernatant isolated from cultures ofburyrylcholinesterase variant library clones. Alternatively, thebutyrylcholinesterase variant can be purified prior to being contactedby the subsurate. Appropriate medium conditions in which to contact acocaine-based substrate with a butyrylcholinesterase variant of theinvention are readily determined by those skilled in the art. Forexample, 100 μM cocaine in 10 mM Ths at pH 7.4 can be contacted with abutyrylcholinesterase variant at 37° C. As described below,butyrylcholinesterase variants from culture supernatants can further beimmobilized using a capture agent, such as an antibody prior to beingcontacted with a substrate, which allows for removal of culturesupernatant components and enables contacting of the immobilizedvariants with substrate in the absence of contaminants. Followingcontacting of a butyrylcholinesterase variant of the invention with acocaine-based substrate, cocaine hydrolysis activity can be measured bya variety of methods known in the art and described herein, for example,by high-performance liquid chromatography or the isotope tracer cocainehydrolysis assay.

The invention also provides a method of treating cocaine overdose aswell as cocaine addiction in an individual by administering atherapeutically effective amount of the butyrylcholinesterase variant.The dosage of a butyrylcholinesterase variant required to be effectivedepends, for example, on whether an acute overdose or chronic addictionis being treated, the route and form of administration, the potency andbio-active half-life of the molecule being administered, the weight andcondition of the individual, and previous or concurrent therapies. Theappropriate amount considered to be an effective dose for a particularapplication of the method can be determined by those skilled in the art,using the teachings and guidance provided herein. For example, theamount can be extrapolated from in vitro or in vivobutyrylcholinesterase assays described herein. One skilled in the artwill recognize that the condition of the individual needs to bemonitored throughout the course of treatment and that the amount of thecomposition that is administered can be adjusted accordingly.

For treating cocaine-overdose, a therapeutically effective amount of abutyrylcholinesterase variant of the invention can be, for example,between about 0.1 mg/kg to 0.15 mg/kg body weight, for example, betweenabout 0.15 mg/kg to 0.3 mg/kg, between about 0.3 mg/kg to 0.5 mg/kg orpreferably between about 1 mg/kg to 5 mg/kg, depending on the treatmentregimen. For example, if a butyrylcholinesterase variant is administeredto an individual symptomatic of cocaine overdose a higher one-time doseis appropriate, while an individual symptomatic of chronic cocaineaddiction may be administered lower doses from one to several times aday, weekly, monthly or less frequently. Similarly, formulations thatallow for timed-release of a butyrylcholinesterase variant would providefor the continuous release of a smaller amount of abutyrylcholinesterase variant to an individual treated for chroniccocaine addiction. It is understood, that the dosage of abutyrylcholinesterase variant has to be adjusted based on the catalyticactivity of the variant, such that a lower dose of a variant exhibitingsignificantly enhanced cocaine hydrolysis activity can be administeredcompared to the dosage necessary for a variant with lower cocainehydrolysis activity.

A butyrylcholinesterase variant can be delivered systemically, such asintravenously or intraarterially. A butyrylcholinesterase variant can beprovided in the form of isolated and substantially purified polypeptidesand polypeptide fragments in pharmaceutically acceptable formulationsusing formulation methods known to those of ordinary skill in the art.These formulations can be administered by standard routes, including forexample, topical, transdermal, intraperitoneal, intracranial,intracerebroventricular, intracerebral, intravaginal, intrauterine,oral, rectal or parenteral (e.g., intravenous, intraspinal, subcutaneousor intramuscular) routes. In addition, a butyrylcholinesterase variantcan be incorporated into biodegradable polymers allowing for sustainedrelease of the compound useful for treating individuals symptomatic ofcocaine addiction. Biodegradable polymers and their use are described,for example, in detail in Brem et al., Neurosurg. 74:441–446 (1991),which is incorporated herein by reference.

A butyrylcholinesterase variant can be administered as a solution orsuspension together with a pharmaceutically acceptable medium. Such apharmaceutically acceptable medium can be, for example, water, sodiumphosphate buffer, phosphate buffered saline, normal saline or Ringer'ssolution or other physiologically buffered saline, or other solvent orvehicle such as a glycol, glycerol, an oil such as olive oil or aninjectable organic ester. A pharmaceutically acceptable medium canadditionally contain physiologically acceptable compounds that act, forexample, to stabilize or increase the absorption of thebutyrylcholinesterase variant. Such physiologically acceptable compoundsinclude, for example, carbohydrates such as glucose, sucrose ordextrans; antioxidants such as ascorbic acid or glutathione; chelatingagents such as EDTA, which disrupts microbial membranes; divalent metalions such as calcium or magnesium; low molecular weight proteins; lipidsor liposomes; or other stabilizers or excipients.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions such as the pharmaceuticallyacceptable mediums described above. The solutions can additionallycontain, for example, buffers, bacteriostats and solutes which renderthe formulation isotonic with the blood of the intended recipient. Otherformulations include, for example, aqueous and non-aqueous sterilesuspensions which can include suspending agents and thickening agents.The formulations can be presented in unit-dose or multi-dose containers,for example, sealed ampules and vials, and can be stored in alyophilized condition requiring, for example, the addition of thesterile liquid carrier, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules and tablets of the kind previously described.

The butyrylcholinesterase variant of the invention can further beutilized in combination therapies with other therapeutic agents.Combination therapies that include a butyrylcholinesterase variant canconsist of formulations containing the variant and the additionaltherapeutic agent individually in a suitable formulation. Alternatively,combination therapies can consist of fusion proteins, where thebutyrylcholinesterase variant is linked to a heterologous protein, suchas a therapeutic protein.

The butyrylcholinesterase variant of the invention also can be deliveredto an individual by administering an encoding nucleic acid for thepeptide or variant. The encoding nucleic acids for thebutyrylcholinesterase variant of the invention are useful in conjunctionwith a wide variety of gene therapy methods known in the art fordelivering a therapeutically effective amount of the polypeptide orvariant. Using the teachings and guidance provided herein, encodingnucleic acids for a butyrylcholinesterase variant can be incorporatedinto a vector or delivery system known in the art and used for deliveryand expression of the encoding sequence to achieve a therapeuticallyeffective amount. Applicable vector and delivery systems known in theart include, for example, retroviral vectors, adenovirus vectors,adenoassociated virus, ligand conjugated particles and nucleic acids fortargeting, isolated DNA and RNA, liposomes, polylysine, and celltherapy, including hepatic cell therapy, employing the transplantationof cells modified to express a butyrylcholinesterase variant, as well asvarious other gene delivery methods and modifications known to thoseskilled in the art, such as those described in Shea et al., NatureBiotechnology 17:551–554 (1999), which is incorporated herein byreference.

Specific examples of methods for the delivery of a butyrylcholinesterasevariant by expressing the encoding nucleic acid sequence are well knownin art and described in, for example, U.S. Pat. No. 5,399,346; U.S. Pat.Nos. 5,580,859; 5,589,466; 5,460,959; 5,656,465; 5,643,578; 5,620,896;5,460,959; 5,506,125; European Patent Application No. EP 0 779 365 A2;PCT No. WO 97/10343; PCT No. WO 97/09441; PCT No. WO 97/10343, all ofwhich are incorporated herein by reference. Other methods known to thoseskilled in the art also exist and are similarly applicable for thedelivery of a butyrylcholinesterase variant by expressing the encodingnucleic acid sequence.

In addition to the treatment of cocaine-induced conditions such ascocaine overdose or cocaine addiction, a butyrylcholinesterase can alsobe administered prophylactically to avoid the onset of a cocaineoverdose upon subsequent entry of cocaine into the bloodstream. It isfurther contemplated that a butyrylcholinesterase variant exhibitingincreased cocaine hydrolysis activity of the invention can havediagnostic value by providing a tool for efficiently determining thepresence and amount of a cocaine-induced substance in a medium.

It is understood that modifications that do not substantially affect theactivity of the various embodiments of this invention are also includedwithin the definition of the invention provided herein. Accordingly, thefollowing examples are intended to illustrate but not limit the presentinvention.

EXAMPLE I A Butyrylcholinesterase Variant with Increased CocaineHydrolysis Activity

This example describes the discovery and characterization of thebutyrylcholinesterase variant designated SEQ ID NO: 2, in which Alanine(A) at amino acid position 328 of human butyrylcholinesterase isreplaced with Tryptophan (W). The A328W butyrylcholinesterase variantdesignated SEQ ID NO: 2 exbibits a 15-fold increase in cocainehydrolysis activity compared to human butyrylcholinesterase.

Structural Modeling of Cocaine in the Active Site of HumanButyrylcholinesterase

In order to determine amino acid residues important for cocainehydrolysis activity, cocaine was docked into the active site ofbutyrylcholinesterase with the FlexiDock program (Tripos Inc., St.Louis, Mo.) in Sybyl 6.4 software on a Silicone Graphics Octanecomputer. Flexidock allows docking of ligands into protein active sites,allowing the user to define bonds which are flexible during the dockingprocess. The user must identify the starting conformation and positionthe interacting faces of the protein-ligand.

The structures of (−)-cocaine and (+)-cocaine were retrieved from theCambridge Structural Database where its code-names are COCAIN10 andCOCHCL. The HCl molecule was deleted from COCHCL so that allcomputations were done with the base form of cocaine. Before theFlexiDock program was run, cocaine was manually aligned withbutyrylcholine in the model of human butyrylcholinesterase as describedby Harel et al., Proc. Natl. Acad. Sci. USA, 89: 10827–10831 (1992).Manual alignment was performed so that the tropane ring of cocaine facedthe Tryptophan residue (W) at amino acid position 82 ofbutyrylcholinesteflse, the carboxyl group of the benzoic ester ofcocaine was within 1.5 Å of the Serine (S) residue at amino acidposition 198 of butyrylcholinesterase, and the benzene ring of cocainewas in the acyl binding pocket of butyrylcholinesterase. In theFlexiDock the binding pocket was defined as all amino acids within 4 Åof butyrylcholine. After defining the binding pocket, the butyrylcholinemolecule was extracted. All atoms in the binding pocket, except atoms inrings and double bonded atoms, were defined as rotatable, thus yielding124 rotatable bonds in butyrylcholinesterase and 7 rotatable bonds incocaine.

Mutagenesis of Human Butyrylcholinesterase and Expression of aButyrylcholinesterase Variant.

Based on the FlexiDock modeling of cocaine into the active site of thehuman butyrylcholinesterase molecule, amino acids that interfere withbinding were selected for mutagenesis.

Thirty-four variants were prepared using PCR-site directed mutagenesisof human butyrylcholinesterase DNA performed utilizing Pfu polymerase(Stratagene, La Jolla, Calif.). Three oligonucleotide primers were usedto perform the mutagenesis. The mutagenesis primers were used at thesame time as a general primer such as the SP6 promoter sequencing primer(MBI Fermentas, Amherst, N.Y.) to amplify one end of thebutyryicholinesterase cDNA. The following primers were used to preparethe A328W mutant: A328W antisense 5′ATAGACTAAAAACCATGTCCCTTCATC 3′ (SEQID NO: 29); T7 old sense 5′TAATACGACTCACTATAGGG 3′ (SEQ ID NO: 30); andSP6 antisense 5′ATTTAGGTGACACTATAG 3′ (SEQ ID NO: 31). The A328W primerspans 27 nucleotides and contains the A328W mutation in the middle ofthe primer. The PCR reaction products (megaprimers) were cleaned onQuiaQuick PCR (Qiagen, Santa Clarita, Calif.) according to themanufacturer's protocol to remove excess primers. The cleanedmegaprimers were extended in a second 2CR reaction to generate thecomplete 1.8 kb coding sequence of each of the 34 variants.

The 1.8-kb fragments constituting the butyrylcholinesterase variantswere cloned into the plasmid pGS and resequenced to make sure thedesired mutation was present. The plasmid pGS is identical with pRc/CMV(Invitrogen, Carlsbad, Calif.) except that the Neo gene has beenreplaced by rat glutamine synthetase.

To express the thirty-four butyrylcholinesterase variants in mammaliancell lines, thirty-four stable Chinese Hamster Ovary (CHO) cell linesexpressing a butyrylcholinesterase variant were made. Transfection ofCHO-KI (No. CCL 61; American Type; Fisher Scientific Co., Pittsburgh,Pa.) cells by calcium phosphate precipitation was followed by selectionof colonies in glutamine-free, serum-free medium Ultraculture containing50 μM methionine sulfoximine (BioWhittaker, Inc., Walkersville, Md.).Colonies expressing the highest levels of butyrylcholinesterase activitywere expanded. A second plasmid that carries the COLQ gene, whichencodes the proline rich attachment domain, was transfected into each ofthe CHO-KI cell lines to allow butyrylcholinesterase to form tetramers,which are more stable.

The secreted butyrylcholinesterase variants were collected from theexpanded cell lines. For collection of large volumes of each secretedbutyrylcholinesterase variant, cells in 1-liter roller bottles were fedevery 2 to 3 days with 100 ml of Ultraculture containing 25 μMmethionine sulfoximine followed by 100 ml of Dulbecco's modified Eagle'smedium and Ham's F12 50:50 mix without L-glutamine (Mediatech, Herndon,Va.; Fisher Scientific Co., Pittsburgh, Pa.). The amount of secretedbutyrylcholinesterase variant is about 1 mg per liter. Twenty liters ofculture medium were collected for each of the thirty-four variants overa period of months and stored sterile at 4° C. during the collectionperiod.

Purification and Characterization of the Butyrylcholinesterase Variants

To purify the butyrylcholinesterase variants, the culture mediumcorresponding to each variant was filtered through Whatman #1 filterpaper (Whatman Inc., Clifton, N.J.) on a Buchner funnel. The filtratewas poured through a chromatography column (XK50/30, Pharmacia Biotech,Piscataway, N.J.) packed with 100ml of affinity gelprocainamide-Sepharose 4B. The butyrylcholinesterase variants stick tothe affinity gel during loading so that 20 mg of enzyme that waspreviously in 20 liters was concentrated in 100 ml of affinity gel. Theaffinity gel was subsequently washed with 3M sodium chloride in 20 mMpotassium phosphate pH 7.0 and 1 mM EDTA to elate contaminatingproteins. Next the affinity gel was washed with buffer containing 20 mMpotassium phosphate and 1 mM EDTA pH 7.0 to reduce the ionic strength.Finally, the butyrylcholinesterase variants were eluted with 250 ml of0.2M procainamide in buffer.

To further purify the butyrylcholinesterase variants and remove theprocainamide a second purification step was performed. Thebutyrylcholinesterase variants recovered in the first purification stepwere diluted 10-fold with buffer (20 mM TrisCl, 1 mM EDTA pH 7.4) toreduce the ionic strength to about 0.02M. The diluted enzyme was loadedonto a column containing 400 ml of the weak anion exchanger DE52(Whatman, Clifton, N.J.). At this low ionic strength thebutyrylcholinesterase variant sticks to the ion exchange gel. Afterloading was complete the column was washed with 2 liters of buffercontaining 20 mM TrisCl and 1 mM EDTA pH 7.4 until the absorbency of theeluant at 280 nm was nearly zero, indicating that the procainamide hadwashed off. Subsequently, the butyrylcholinesterase variants were elutedfrom the column with a salt gradient from 0 to 0.2M NaCl in 20 mM TrisClpH7.4. Following the elution of the butyrylcholinesterase variants 10 mlfractions were collected for each variant using a fraction collector.Activity assays were performed to identify the peak containingbutyrylcholinesterase variant SDS gel electrophoresis was performed todetermine the purity of each butyrylcholinesterase variant, which wasdetermined to be approximately 90%.

The thirty-four purified butyrylcholinestesterase variants were assayedfor their ability to hydrolyze cocaine. The assay measured the affinityof (−)-cocaine for the butyrylcholinesterase variants and the maximalrate of hydrolysis of (−)-cocaine for each variant. Enzyme-catalyzedhydrolysis of cocaine was recorded on a temperature-equilibrated GilfordSpectrophotometer at 240 nm where the difference in molar absorptivitybetween substrate and product was ΔE=6,700M⁻¹ cm⁻¹ as described byGatley, Biochem. Pharmacol. 41:1249–1254 (1991). K_(m) values weredetermined in 0.1M potassium phosphate pH 7.0 at 30° C. for (−)-cocaine.V_(max) values and K_(m) values were calculated using Sigma Plot forMacintosh (Jandel Scientific, San Rafael, Calif.).

Once V_(max) values and K_(m) values were calculated, the number ofactive sites in each butyrylcholinesterase preparation was determined.The titration of active sites was performed with chlorpyrifos oxon(MET-674B, Chem Service, West Chester, Pa.), an inhibitor ofbutyrylcholinesterase. One molecule of chlorpyrifos oxon binds andinhibits one molecule of butyrylcholinesterase, which allows forcalculation of the number of active sites. Based on the number of activesites, the k_(cat) value for each variant was calculated (Table 3).Thirty-four variants were tested for cocaine binding or cocainehydrolysis (Table 4). One variant, A328W, was determined to have 15times faster cocaine hydrolysis activity compared to wild-typebutyrylcholinesterase.

TABLE 3 Binding constant (K₁ and K_(m)) and hydrolysis rate (k_(cat))for human butyrylcholinesterase and mutants K₁ (μM) K_(m) (μM) k_(cat)(min⁻¹) wild-type 11 14 3.9 D70G 201 D70N 490 G117H 440 G117K 300 Q119H34 Q119Y 56 2.0 T120F 97 E197D 40 E197G 37 E197Q 17 0.1 L286A 8.5 L286H24 V288F 17 1.0 V288H 55 A328F 21 24 5.8 A328G 18 A328H 27 A328I 11 0.5A328W 10 37.2 A328Y 9 10.2 F329A 128 2.7 F329S 41 1.9 Y332A 240 Y332F 22G439A 7 N68Y/Q119Y/A277W 60 1.7 Q119Y/V288F/A328Y 33 2.3

TABLE 4 Mutants tested for cocaine binding or hydrolysis (34 pluswild-type) Mutant Cocaine Binding or Cocaine Hydrolysis wild-type Ki =11 μM D70G Ki = 201 μM D70N Ki = 490 μM G115A no activity G116F noactivity G116W no activity G117H Ki = 440 μM Q119H Ki = 34 μM Q119Y nota cocaine hydrolase T120F not a cocaine hydrolase E197D Ki = 40 μM E197GKi = 37 μM E197Q Not a cocaine hydrolase S224Y No activity L286A Ki = 24μM L286H Not a cocaine hydrolase L286W Not a cocaine hydrolase V288F Nota cocaine hydrolase V288H Ki = 55 μM V288W Not a cocaine hydrolase A328FNot a cocaine hydrolase A328G Not a cocaine hydrolase A328H Not acocaine hydrolase A328I Not a cocaine hydrolase A328W Hydrolyzes cocaine15 times faster than wild-type A328Y Hydrolyzes cocaine 4 times fasterthan wild-type F329A Not a cocaine hydrolase F329S kcat is faster thanwild type Y332F Ki = 22 μM G439A Ki = 7 μM G439L No cocaine hydrolysisactivity N68Y/Q119Y/A277W Not a cocaine hydrolase Q119Y/V288F/A328Y Nota cocaine hydrolase

EXAMPLE II Development of a Cocaine Hydrolysis Assay

This example describes the development of a cocaine hydrolysis assaythat permits the efficient analysis of hundreds of butyrylcholinesterasevariants simultaneously.

Development of an Isotope Tracer Cocaine Hydrolysis Assay.

For the purpose of validating new cocaine hydrolysis assays,butyrylcholinesterase hydrolysis of cocaine was first measured asdescribed previously (Xie et al., Mol. Pharmacol. 55:83–91 (1999)),using high-performance liquid chromatography (HPLC). Briefly, reactionscontaining 100 μM cocaine in 10 mM Tris, pH 7.4 were initiated by theaddition of horse butyrylcholinesterase (ICN Pharmaceuticals, Inc.,Costa Mesa, Calif.) and incubated 2–4 hours at 37° C. Following theincubation, the pH was adjusted to 3, and the sample was filtered.Subsequently, the sample was applied to a Hypersil ODS-C 18 reversedphase column (Hewlett Packard, Wilmington, Del.) previously equilibratedwith an 80:20 mixture of 0.05 M potassium phosphate, pH 3.0 andacetonitrile. The isocratic elution of cocaine, benzoylecognine, andbenzoic acid was quantitated at 220 nm. Measurement of the formation ofecognine methyl ester and benzoic acid was dependent both on the amountof butyrylcholinesterase in the reaction and on the time of reaction.

At the conclusion of the isotope tracer assay, an aliquot of thereaction mix is acidified in order to take advantage of the solubilitydifference between the product and the substrate at pH 3.0. At pH 3.0,[3H]-benzoic acid (pKa=4.2) is soluble in a scintillation cocktailconsisting of 2,5-diphenyloxazole (PPO) and[1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene](POPOP)(PPO-dimethyl-POPOP scintillation fluor, Research Products InternationalCorp., Mt. Prospect, Ill) while [3H]-cocaine is not. The signalgenerated by acidified reaction mixture from enzyme blanks was less than2% of the total dpm placed in the fluor, consistent with cocaine beinginsoluble in PPO-dimethyl-POPOP.

The isotope tracer cocaine hydrolysis assay was validated by directcomparison with the established HPLC assay and the accuracy of theisotope assay was demonstrated by determining the K_(m) value for horsebutyrylcholinesterase. The rate of cocaine hydrolysis, determined bymeasuring the rate of formation of benzoic acid was quantitated both byHPLC and the isotope tracer assay in reactions containing variableamounts of butyrylcholinesterase. Formation of [³H]-benzoic acid wasdependent on the length of assay incubation and on the amount ofbutyrylcholinesterase added. Good correlation between the establishedHPLC assay and the isotope tracer assay was observed, as demonstrated byplotting the quantitation of benzoic acid formation measured by HPLCversus the benzoic acid formation measured in the isotope assay (seeFIG. 5A; r²=0.979). To demonstrate the precision and sensitivity of theisotope assay the amount of cocaine was varied and the K_(m) wasdetermined using the Lineweaver-Burk double-reciprocal plot of cocainehydrolysis by horse butyrylcholinesterase depicted in FIG. 5B. Velocitywas calculated as cpm benzoic acid formed×10⁻⁵ following a 2 hourincubation at 37° C. Based on these data the K_(m) for cocainehydrolysis is approximately 37.6 μM (×intercept=−l/K_(m)), which is inclose agreement with previously published values of 38 μM (Gatley,supra, 1991) and 45±5 μM (Xie et al., supra, 1999) for horsebutyrylcholinesterase.

Immobilization of Active Butyrylcholinesterase.

The supernatants isolated from each of the butyrylcholinesterase variantlibrary clones contains variable butyrylcholinesterase enzymeconcentrations. Consequently, the cocaine hydrolysis activity measuredfrom equal volumes of culture supernatants from distinctbutyrylcholinesterase variant clones reflects the expression level aswell as the enzyme activity. In order to be able to compare equal enzymeconcentrations and more rapidly identify variants with the desiredactivity, butyrylcholinesterase from culture supernatants areimmobilized using a capture reagent, such as an antibody, that issaturated at low butyrylcholinesterase concentrations as describedpreviously by Watkins et al., Anal. Biochem. 253: 37–45 (1997). As aresult, butyrylcholinesterase from dilute samples is concentrated anduniform quantities of different butyrylcholinesterase variant clones areimmobilized, regardless of the initial concentration ofbutyrylcholinesterase in the culture supernatant. Subsequently, unboundbutyrylcholinesterase and other culture supernatant components thatpotentially interfere with the assay (such as unrelated serum orcell-derived proteins with significant esterase activity) are washedaway and the activity of the immobilized butyrylcholinesterase isdetermined by measuring the formation of benzoic acid as describedabove.

To assess the efficiency of the above assay, efficient capture of humanbutyrylcholinesterase, as well as a truncated soluble monomeric form ofhuman butyrylcholinesterase (Blong et al., Biochem. J. 327: 747–757(1997)), was demonstrated in a microtiter format using a commerciallyavailable rabbit anti-human cholinesterase polyclonal antibody (DAKO,Carpinteria, Calif.) (FIG. 6). In order to determine the optimalconditions for capturing butyrylcholinesterase a microtiter plate wascoated with increasing quantities of rabbit anti-butyrylcholinesterase,was blocked, and incubated with varying amounts of culture supernatant.The amount of active butyrylcholinesterase captured was determinedcalorimetrically using an assay that measures butyrylthiocholinehydrolysis at 405 nm in the presence of dithiobisnitrobenzoic acid (Xieet al., supra, 1999). Subsequently, the butyrylcholinesterase activitycaptured from dilutions of culture supernatants from cells expressingeither the wild-type human butyrylcholinesterase or the monomerictruncated version was measured. The rabbit anti-butyrylcholinesterasecapture antibody was saturated by the butyrylcholinesterase present in25 μl of culture supernatant with greater butyrylcholinesterase activitybeing captured from supernatant containing the full length wild-typeform of the enzyme (FIG. 6, compare filled circles with open circles).Unbound material was removed by washing with 100 mM Tris, pH 7.4 and theamount of active butyrylcholinesterase captured was quantitated bymeasuring butyrylthiocholine hydrolysis. Butyrylcholinesterase isexpressed in culture supernatants at quantities sufficient to saturate apolyclonal anti-butyrylcholinesterase antibody on a microtiter plate. Inaddition, the captured enzyme is active, as demonstrated by thehydrolysis of butyrylthiocholine.

Measurement of Cocaine Hydrolysis with Isotope Tracer Assay andImmobilized Butyrylcholinesterase

The optimal conditions for immobilization of activebutyrylcholinesterase are used in conjunction with the cocaine isotopetracer assay to measure the cocaine hydrolysis activity in a microtiterformat. The assay is characterized by determining the K_(m) for cocainehydrolysis activity, as described above. At least three approaches areused to either increase the assay sensitivity or the assay signal.

First, longer assay incubation times that proportionately increase thesignal can be used. Second, the sensitivity of the assay can be enhancedby increasing the specific activity of the radiolabeled cocainesubstrate. Third, a previously identified butyrylcholinesterase mutantwhich is 4-fold more efficient for cocaine hydrolysis can used (Xie etal., supra, 1999), which in conjunction with doubling the assayincubation time and increasing the specific activity of the cocaine10-fold, can increase the assay signal about 80-fold.

EXAMPLE III Synthesis and Characterization of ButyrylcholinesteraseVariant Libraries

This example describes the synthesis and characterization ofbutyrylcholinesterase variant libraries expressed in mammalian cells.

In order to facilitate the synthesis of libraries ofbutyrylcholinesterase variants, DNA encoding wild-type humanbutyrylcholinesterase, a truncated, enzymatically active, monomericversion of human butyrylcholinesterase, and the A328Y mutant thatdisplays a four-fold increased cocaine hydrolysis activity are clonedinto a modified doublelox targeting vector, using unique restrictionsites. In preliminary assays the wild-type human butyrylcholinesterasewas captured more efficiently and, therefore, serves as the initial DNAtemplate for the synthesis of libraries of butyrylcholinesterasevariants.

Synthesis of Focused Libraries of Butyrylcholinesterase Variants byCodon-based Mutagenesis.

A variety of information can be used to focus the synthesis of theinitial libraries of butyrylcholinesterase variants to discreet regions.For example, butyrylcholinesterase and Torpedo acetylcholinesterase(AChE) share a high degree of homology (53% identity). Furthermore,residues 4 to 534 of Torpedo AChE can be aligned with residues 2 to 532of butyrylcholinesterase without deletions or insertions. The catalytictriad residues (butyrylcholinesterase residues Ser198, Glu325, andHis438) and the intrachain disulfides are all in the same positions. Dueto the high degree of similarity between these proteins, a refined 2.8-Åx-ray structure of Torpedo AChE (Sussman et al., Science 253: 872–879(1991)) has been used to model butyrylcholinesterase structure (Harel etal., supra, 1992)).

Studies with cholinesterases have revealed that the catalytic triad andother residues involved in ligand binding are positioned within a deep,narrow, active-site gorge rich in hydrophobic residues (reviewed inSoreq et al., Trends Biochem. Sci. 17:353–358 (1992)). The sites ofseven focused libraries of butyrylcholinesterase variants (FIG. 2,underlined residues) were selected to include amino acids determined tobe lining the active site gorge (FIG. 2, hydrophobic active site gorgeresidues are shaded).

In addition to the structural modeling of butyrylcholinesterase,butyrylcholinesterase biochemical data was integrated into the librarydesign process. For example, characterization of naturally occurringbutyrylcholinesterases with altered cocaine hydrolysis activity andsite-directed mutagenesis studies provide information regarding aminoacid positions and segments important for cocaine hydrolysis activity(reviewed in Schwartz et al., Pharmac. Ther. 67: 283–322(1995)).Moreover, comparison of sequence and cocaine hydrolysis data ofbutyrylcholinesterases from different species can also provideinformation regarding regions important for cocaine hydrolysis activityof the molecule based on comparison of the cocaine hydrolysis activitiesof these butyrylcholinesterases. The A328Y mutant described above ispresent in the library corresponding to SEQ ID NO: 8 and serves as acontrol to demonstrate the quality of the library synthesis andexpression in mammalian cells as well as the sensitivity of themicrotiter-based cocaine hydrolysis assay.

The seven regions of butyrylcholinesterase selected for focused librarysynthesis (summarized in Table 2) span residues that include the 8aromatic active site gorge residues (W82, W112, Y128, W231, F329, Y332,W430 and Y440) as well as two of the catalytic triad residues. Theintegrity of intrachain disulfide bonds, located between ⁶⁵Cys-⁹²Cys,²⁵²Cys-²⁶³Cys, and ⁴⁰⁰Cys-⁵¹⁹Cys is maintained to ensure functionalbutyrylcholinesterase structure. In addition, putative glycosylationsites (N-X-S/T) located at residues 17, 57, 106, 241, 256, 341, 455,481, 485, and 486 also are avoided in the library syntheses. In total,the seven focused libraries span 79 residues, representing approximately14% of the butyrylcholinesterase linear sequence, and result in theexpression of about 1500 distinct butyrylcholinesterase variants.

Libraries of nucleic acids corresponding to the seven regions of humanbutyrylcholinesterase to be mutated are synthesized by codon-basedmutagenesis, as described above and as depicted schematically in FIG. 7.Briefly, multiple DNA synthesis columns are used for synthesizing theoligonucleotides by β-cyanoethyl phosphoramidite chemistry, as describedpreviously by Glaser et al., supra, 1992. In the first step,trinucleotides encoding for the amino acids of butyrylcholinesterase aresynthesized on one column while a second column is used to synthesizethe trinucleotide NN(G/T), where N is a mixture of dA, dG, dC, and dTcyanoethyl phosphoramidites. Using the trinucleotide NN(G/T) results inthorough mutagenesis with minimal degeneracy, accomplished through thesystematic expression of all twenty amino acids at every position.

Following the synthesis of the first codon, resins from the two columnsare mixed together, divided, and replaced in four columns. By addingadditional synthesis columns for each codon and mixing the column resinsin the manner illustrated in FIG. 7, pools of degenerateoligonucleotides will be segregated based on the extent of mutagenesis.The resin mixing aspect of codon-based mutagenesis makes the processrapid and cost-effective because it eliminates the need to synthesizemultiple oligonucleotides. In the present study, the pooi ofoligonucleotides encoding single amino acid mutations is used tosynthesize focused butyrylcholinesterase libraries.

The oliganucleotides encoding the butyrylcholinesterase variantscontaining a single amino acid mutation is cloned into the doubleloxtargeting vector using oligonucleotide-directed mutagenesis (Kunkel,supra, 1985). To improve the mutagenesis efficiency and diminish thenumber of clones expressing wild-type butyrylcholinesterase, thelibraries are synthesized in a two-step process. In the first step, thebutyrylcholinesterase DNA sequence corresponding to each library site isdeleted by hybridization mutagenesis. In the second step,uracil-containing single-stranded DNA for each deletion mutant, onedeletion mutant corresponding to each library, is isolated and used astemplate for synthesis of the libraries by oligonucleotide-directedmutagenesis. This approach has been used routinely for the synthesis ofantibody libraries and results in more uniform mutagenesis by removingannealing biases that potentially arise from the differing DNA sequenceof the mutagenic oligonucleotides. In addition, the two-step processdecreases the frequency of wild-type sequences relative to the variantsin the libraries, and consequently makes library screening moreefficient by eliminating repetitious screening of clones encodingwild-type butyrylcholinesterase.

The quality of the libraries and the efficiency of mutagenesis ischaracterized by obtaining DNA sequence from approximately 20 randomlyselected clones from each library. The DNA sequences demonstrate thatmutagenesis occurs at multiple positions within each library and thatmultiple amino acids were expressed at each position. Furthermore, DNAsequence of randomly selected clones demonstrates that the librariescontain diverse clones and are not dominated by a few clones.

Optimization of Transfection Parameters for Site-specific Integration

Optimization of transfection parameters for Cre-mediated site-specificintegration was achieved utilizing Bleomycin Resistance Protein (BRP)DNA as a model system.

Cre recombinase is a well-characterized 38-kDa DNA recombinase (Abremskiet al., Cell 32:1301–1311 (1983)) that is both necessary and sufficientfor sequence-specific recombination in bacteriophage P1. Recombinationoccurs between two 34-base pair loxP sequences each consisting of twoinverted 13-base pair recombinase recognition sequences that surround acore region (Sternberg and Hamilton, J. Mol. Biol. 150:467–486 (1981a);Sternberg and Hamilton, J. Mol. Biol., 150:487–507 (1981b)). DNAcleavage and strand exchange occurs on the top or bottom strand at theedges of the core region. Cre recombinase also catalyzes site-specificrecombination in eukaryotes, including both yeast (Sauer, Mol. Cell.Biol. 7:2087–2096 (1987)) and mammalian cells (Sauer and Henderson,Proc. Natl. Acad. Sci. USA, 85:5166–5170 (1988); Fukushige and Sauer,Proc. Natl. Acad. Sci. U.S.A. 89:7905–7909 (1992); Bethke and Sauer,Nuc. Acids Res., 25:2828–2834 (1997)).

Calcium phosphate transfection of 13–1 cells was previously demonstratedto result in targeted integration in 1% of the viable cells plated(Bethke and Sauer, Nuc. Acids Res., 25:2828–2834 (1997)). Therefore,initial studies were conducted using calcium phosphate to transfect 13–1cells with 4 μg pBS185 and 10, 20, 30, or 40 μg of pBS397-fl(+)/BRP. Thetotal level of DNA per transfection was held constant using unrelatedpBluescript II KS DNA (Stratagene; La Jolla, Calif.), and transformantswere selected 48 hours later by replating in media containing 400 μg/mlgeneticin. Colonies were counted 10 days later to determine theefficiency of targeted integration. Optimal targeted integration wastypically observed using 30 μg of targeting vector and 4 μg of Crerecombinase vector pBS185, consistent with the 20 μg targeting vectorand 5 μg of pBS185 previously reported (Bethke and Sauer, Nuc. AcidsRes., 25:2828–2834 (1997)). The frequency of targeted integrationobserved was generally less than 1%. Despite the sensitivity of thecalcium phosphate methodology to the amount of DNA used and the bufferpH, targeted integration efficiencies observed were sufficient toexpress the protein libraries.

As shown in Table 5, several cell lines as well as other transfectionmethods were also characterized. As disclosed herein, Flp recombinasealso can used to target insertion of exogenous DNA into a particularsite in the genome as described by Dymecki, supra,1996. The target sitefor Flp recombinase consists of 13 base-pair repeats separated by an 8base-pair spacer: 5′-GAAGTTCCTATTC [TCTAGAAA]GTATAGGAACTTC- 3′ (SEQ IDNO: 24). Briefly, variant libraries corresponding to the region ofbutyrylcholinesterase corresponding to amino acids 277–289 (SEQ ID NO:13) of butyryicholinesterase (shown as region 5 in Table 2) weretransfected into mammalian cells using flp recombinase and the 293T cellline. Table 5 shows the butyryicholinesterase variants S285G, P285Q andP285S that were identified and characterized using the methods describedherein utilizing Flp recombinase and the 293T human cell line.

In general, lipid-mediated transfection methods are more efficient thanmethods that alter the chemical environment, such as calcium phosphateand DEAE-dextran transfection. In addition, lipid-mediated transfectionsare less affected by contaminants in the DNA preparations, saltconcentration, and pH and thus generally provide more reproducibleresults (Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413–7417(1987)). Consequently, a formulation of the neutral lipid dioleoylphosphatidylethanolamine and a cationic lipid, termed GenePORTERtransfection reagent (Gene Therapy Systems; San Diego, Calif.), wasevaluated as an alternative transfection approach. Briefly,endotoxin-free DNA was prepared for both the targeting vectorpBS397-fl(+)/BRP and the Cre recombinase vector pBS185 using theEndoFree Plasmid Maxi kit (QIAGEN; Valencia, Calif.). Next, 5 μg pBS185and varying amounts of pBS397-fl(+)/BRP were diluted in serum-freemedium and mixed with the GenePORTER transfection reagent. The DNA/lipidmixture was then added to a 60–70% confluent monolayer of 13–1 cellsconsisting of approximately 5×10⁵ cells/100-mm dish and incubated at 37°C. Five hours later, fetal calf serum was added to 10%, and the next daythe transfection media was removed and replaced with fresh media.

Transfection of the cells with variable quantities of the targetingvector yielded targeted integration efficiencies ranging from 0.1% to1.0%, with the optimal targeted integration efficiency observed using 5μg each of the targeting vector and the Cre recombinase vector.Lipid-based transfection of the 13–1 host cells under the optimizedconditions resulted in 0.5% targeted integration efficiency beingconsistently observed. A 0.5% targeted integration is slightly less thanthe previously reported 1.0% efficiency (Bethke and Sauer, Nuc. AcidsRes., 25:2828–2834 (1997)), and is sufficient to express large proteinlibraries and allows expressing libraries of protein variants inmammalian cells.

TABLE 5 Expression of a single butyrylcholinesterase variant per cellusing either stable or transient cell transfection. Cell IntegrationIntegration? Integration? Line Expression Method (PCR) (Activity) NIH3T3Transient N/A N/A Transient, (13-1) (lipid- very low based) activityNIH3T3 Stable Cre Yes No measurable (13-1) recombinase activity CHOTransient N/A N/A Transient, (lipid- measurable based) activity(colorimetric and cocaine hydrolysis) 293 Transient N/A N/A Transient,(lipid- measurable based) activity (colorimetric and cocaine hydrolysis)293 Stable Flp Yes Measurable recombinase activity (colorimetric andcocaine hydrolysis)

These results demonstrate optimization of transfection conditions fortargeted insertion in NlH3T3 13–1 cells. Conditions for a simple,lipid-based transfection method that required a small amount of DNA andgenerated reproducible 0.5% targeting efficiency were established.

Expression of Butyrylcholinesterase Variant Libraries in Mammalian Cells

Each of the seven libraries of butyrylcholinesterase variants aretransformed into a host mammalian cell line using the doubleloxtargeting vector and the optimized transfection conditions describedabove. Following Cre-mediated transformation the host cells are platedat limiting dilutions to isolate distinct clones in a 96-well format.Cells with the butyrylcholinesterase variants integrated in the Cre/loxtargeting site are selected with geneticin. Subsequently, the DNAencoding butyrylcholinesterase variants from 20–30 randomly selectedclones from each library are sequenced and analyzed as described above.Briefly, total cellular DNA is isolated from about 10⁴ cells of eachclone of interest using DNeasy Tissue Kits (Qiagen, Valencia, Calif.).Next, the butyrylcholinesterase gene is amplified using PfuTurbo DNApolymerase (Stratagene; La Jolla, Calif.) and an aliquot of the PCRproduct is then used for sequencing the DNA encodingbutyrylcholinesterase variants from randomly selected clones by thefluorescent dideoxynucleotide termination method (Perkin-Elmer, Norwalk,Conn.) using a nested oligonucleotide primer.

As described previously, the sequencing demonstrates uniformintroduction of the library and the diversity of mammalian transformantsresembles the diversity of the library in the doubleox targeting vectorfollowing transformation of bacteria.

TABLE 6 Identification and characteristics of butyrylcholinesterasevariant with enhanced cocaine hydrolase activity. Clone SequenceRelative V_(max)/K_(m) 5.2.390F Wild-type human BChE 1.00 A328W 13.45.2.258F S287G 4.3 5.2.444F P285Q 3.9 5.2.600F P285S 2.8

As described herein, a library corresponding to region five ofbutyrylcholinesterase was expressed and individual variants werescreened by measuring the hydrolysis of [³H]-cocaine using themicrotiter assay. The catalytic efficiency (V_(max)/K_(m)) of variantswith enhanced activity were characterized using the microtiter assay todetermine their relative K_(m) and V_(max). Three butyrylcholinesterasevariants were identified that have enhanced cocaine hydrolase activity:S287G (SEQ ID NO: 4), P285Q (SEQ ID NO: 6) and P285S (SEQ ID NO: 8).

EXAMPLE IV Characterization of Butyrylcholinesterase Variants thatDisplay Enhanced Cocaine Hydrolysis Activity

This example describes the molecular characterization ofbutyrylcholinesterase variants that display enhanced cocaine hydrolysisactivity in the microtiter assay described below. The cocaine hydrolysisactivity measured in the microtiter assay format is further confirmedusing greater amounts of the butyrylcholinesterase variants of interest.In addition to the microtiter-based assay, the activity of the clones isdemonstrated in solution phase with product formation measured by theHPLC assay to verify the increased cocaine hydrolysis activity of thebutyrylcholinesterase variants and confirm that the enhanced hydrolysisis at the benzoyl ester group.

The kinetic constants for wild-type butyrylcholinesterase and the bestvariants are determined and used to compare the catalytic efficiency ofthe variants relative to wild-type butyrylcholinesterase. K_(m) valuesfor (−)-cocaine are determined at 37° C. V_(max) and K_(m) values arecalculated using Sigma Plot (Jandel Scientific, San Rafael, Calif.). Thenumber of active sites of butyrylcholinesterase is determined by themethod of residual activity using echothiopate iodide or diisopropylfluorophosphates as titrants, as described previously by Masson et al.,Biochemistry 36: 2266–2277 (1997). Alternatively, the number ofbutyrylcholinesterase active sites is estimated using an ELISA toquantitate the mass of butyrylcholinesterase or butyrylcholinesterasevariants present in culture supernatants. Purified humanbutyrylcholinesterase is used as the standard for the ELISA quantitationassay. The catalytic rate constant, k_(cat), is calculated by dividingV_(max) by the concentration of active sites. Finally, the catalyticefficiencies of the best variants are compared to wild-typebutyrylcholinesterase by determining k_(cat)/K_(m) for eachbutyrylcholinesterase variant.

In order to better characterize all the clones expressingbutyrylcholinesterase variants with increased cocaine hydrolysisactivity, the DNA encoding the variants is sequenced. DNA sequencingreveals the precise location and nature of the mutations and thus,quantifies the total number of distinct butyrylcholinesterase variantsidentified. Screening of each library is complete when clones encodingidentical butyrylcholinesterase mutations are identified on multipleoccasions, indicating that the libraries have been screenedexhaustively.

EXAMPLE V Synthesis and Characterization of CombinatorialButyrylcholinesterase Variant Libraries

This example demonstrates synthesis and characterization ofcombinatorial libraries of butyrylcholinesterase variants expressed inmammalian cells.

The beneficial mutations identified from screening libraries ofbutyrylcholinesterase variants containing a single amino acid mutationare combined in vitro to further improve the butyrylcholinesterasecocaine hydrolysis activity. The positive combination of beneficialmutations designated biochemical additivity has been observed onmultiple occasions. For example, the iterative process of increasingantibody affinity in a stepwise fashion through the accumulation andsubsequent combination of beneficial mutations has led to theidentification of antibodies displaying 500-fold enhanced affinity usingvariant libraries containing less than 2,500 distinct variants.Importantly, the principle of biochemical additivity is not restrictedto improving the affinity of antibodies, and has been exploited toachieve improvements in other physical properties, such asthermostability, catalytic efficiency, or enhanced resistance topesticides.

The best mutations identified from screening the seven focusedbutyrylcholinesterase libraries are used to synthesize a combinatoriallibrary. The number of distinct variants in the combinatorial library isexpected to be small, typically a fraction of the number of distinctvariants from the initial libraries. For example, combinatorial analysisof single mutations at eight distinct sites would require a library thatcontains 2⁸, or 256, unique variants. The combinatorial library issynthesized by oligonucleotide-directed mutagenesis, characterized, andexpressed in the mammalian host cell line. Variants are screened andcharacterized as described above. DNA sequencing reveals additivemutations.

EXAMPLE VI Expression and Purification of Butyrylcholinesterase Variants

This example demonstrates the expression in a mammalian cell line andsubsequent purification of butyrylcholinesterase variants.

Clones expressing the most catalytically active butyrylcholinesterasevariants, as well as wild-type butyrylcholinesterase, are used toestablish larger-scale cultures in order to purify quantities of theenzyme necessary for in vivo studies. It is estimated that approximately100 mg each of wild-type butyrylcholinesterase and the optimal variantis required to complete the in vivo toxicity and addiction studies inrats as described below.

The butyrylcholinesterase variants of interest are cloned into thepCMV/Zeo vector (Invitrogen, Carlsbad, Calif.) using unique restrictionsites. The cloning of the variants is verified using restriction mappingand DNA sequencing. Subsequently, the variants are expressed intransfected Chinese Hamster ovary cells CHO Kl (ATCC CCL 61). CHO cellswere selected for expression because butyrylcholinesterase is aglycoprotein and these cells have been previously used for theexpression of recombinant human therapeutic glycoproteins (Goochee etal., Biotechnology 9:1347–1355 (1991); Jenkins and Curling, EnzymeMicrob. Technol. 16:354–364 (1994)) as well as fully active recombinantbutyrylcholinesterase (Masson et al., supra, 1997). Initially, the CHOcells are transiently transfected with all the butyrylcholinesterasevariants to confirm expression of functional butyrylcholinesterase.Subsequently, the cells are stably transfected and clones expressingbutyrylcholinesterase variants are selected using the antibiotic Zeocin(Invitrogen. Carlsbad, Calif.). Colonies are picked with a sterilecotton-tipped stick and transferred to 24-well plates. Thebutyrylcholinesterase expression is measured and the colonies with thehighest activity are further expanded. The kinetic constants of thebutyrylcholinesterase variants are determined to ensure that expressionin CHO cells does not diminish the enzymatic activity compared tobutyrylcholinesterase variants expressed in NIH3T3 cells.

The cells are expanded in T175 flasks and expanded further into multiple3L spinner flasks until approximately 5×10⁸ cells are obtained.Subsequently, the cell lines are transferred to CELL-PHARM System 2000hollow fiber cell culture systems (Unisyn Technologies, Hopkinton,Mass.) for the production and continuous recovery ofbutyrylcholinesterase. The hollow fiber system permits high celldensities to be obtained (10⁸/ml) from which 60–120 ml of concentratedbutyrylcholinesterase is harvested each day. It is anticipated that itrequires one month to produce sufficient quantities ofbutyrylcholinesterase for further evaluation.

The concentrated recombinant butyrylcholinesterase harvested from thehollow fiber systems are purified, essentially as described previously(Masson et al., supra, 1997). The serum-free medium is centrifuged toremove particulates, its ionic strength is reduced by dilution with twovolumes of water, and subsequently, the sample is loaded on aprocainamide Sepharose affinity column. Butyrylcholinesterase is elutedwith procainamide, purified further by ion exchange chromatography andconcentrated. A recombinant butyrylcholinesterase mutant expressed inCHO cells has previously been enriched to 99% purity with over 50%yields using this purification approach (Lockridge et al., Biochemistry36:786–795 (1997)). The enzyme is filter-sterilized through a 0.22-μmmembrane and stored at 4° C. Under these conditions,butyrylcholinesterase retains over 90% of its original activity after 18months (Lynch et al., Toxicology and Applied Pharmacol. 55:83–91 (1999))

EXAMPLE VII Evaluation of Wild-Type Butyrylcholinesterase andButyrylcholinesterase Variants

This example describes the evaluation of wild-type butyrylcholinesteraseand butyrylcholinesterase variants in rat cocaine toxicity andreinforcement models.

Butyrylcholinesterase variants that display increased cocaine hydrolysisactivity in vitro display greater potency for the treatment of cocainetoxicity and addiction in vivo. To characterize thebutyrylcholinesterase variants in vivo, an acute overdose model is usedto measure the potency of butyrylcholinesterase variants for toxicity,while models of reinforcement and discrimination are used to predict thepotency of butyrylcholinesterase variants for the treatment ofaddiction. Although the pharmacokinetics of human butyrylcholinesterasevariants are not expected to be optimal in models, the rat cocainemodels are well characterized and require significantly smallerquantities of purified butyrylcholinesterase than do primate models. Itis anticipated that both wild-type butyrylcholinesterase and thebutyrylcholinesterase variants with increased cocaine hydrolysisactivity display dose-dependent responses. Furthermore, thebutyrylcholinesterase variant optimized for cocaine hydrolysis activityare efficacious at substantially smaller doses than the wild-typebutyrylcholinesterase.

Modification of the Toxicity of Cocaine

The effect of butyrylcholinesterase variants on cocaine toxicity isevaluated as previously described in rat model of overdose by Mets etal., Proc. Nat. Acad. Sci. USA 95:10176–10181 (1998). This model usesco-infusion of catecholamines because variable endogenous catecholaminelevels have been shown to affect cocaine toxicity (Mets et al., LifeSci. 59:2021–2031 (1996)). Infusion of cocaine at 1 mg/kg/min producesLD₅₀=10 mg/kg and LD₉₀=16 mg/kg when the levels of catecholamines arestandardized.

Six groups of six rats each are used in this study. The rats areSprague-Dawley males, weighing 250–275 g upon receipt in the vivarium,which is maintained on a 12 hour light-dark cycle. The rats have foodand water available ad libitum at all times. Prior to treatment the ratsare fitted with femoral arterial and venous catheters and permitted torecover. Subsequently, the rats are treated with varying amounts of thebutyrylcholinesterase variants (0.35, 1.76, or 11.8 mg/kg) or equivalentvolumes of saline 15 minutes prior to the co-infusion of catecholaminesand cocaine (1 mg/kg/min). The infusion is for 16 minutes to deliver theLD₉₀ of cocaine, unless the animals expire sooner. Based on the relativecatalytic efficiencies of wild-type butyrylcholinesterase and thepreviously described catalytic antibody (Mets et al., supra, 1998), itis anticipated that increasing doses of butyrylcholinesterase conferincreased survival rate to the rats relative to the saline controls andthat the highest butyrylcholinesterase dose (11.8 mg/kg) protects allthe animals. A butyrylcholinesterase variant that hydrolyzes cocaine10-fold more efficiently in vitro is expected to confer protection toall of the animals at a lower dose (1 mg/kg, for example).

Modification of the Abuse of Cocaine

The discriminative and reinforcing pharmacological effects of cocaineare believed to most closely reflect the actions of cocaine that embodyabuse of the drug. Therefore, the butyrylcholinesterase variants areevaluated in both cocaine reinforcement and cocaine discriminationmodels in rats.

The rat model of the reinforcing effects of cocaine has been usedextensively to evaluate other potential therapies for cocaine (Koob etal., Neurosci. Lett. 79: 315–320(1987); Hubner and Moreton,Psychopharmacology 105: 151–156 (1991); Caine and Koob, J. Pharmacol.Exp. Ther. 270:209–218 (1994); Richardson et al., Brain Res. 619: 15–21(1993)).

Male Sprague-Dawley rats are maintained as described above. Six operantchambers (Med Associates, St. Albans, Vt.), equipped with a house light,retractable lever, dipper mechanism, red, yellow, and green stimuluslights, and a pneumatic syringe-drive pump apparatus (IITC LifeSciences, Inc., Woodland Hills, Calif.) for drug delivery are interfacedwith an IBM-compatible computer through input and output cards (MedAssociates, Inc., St. Albans, Vt.). The chambers are housed within anair conditioned, sound attenuating cubicle (Med Associates). Customself-administration programs, controlling scheduled contingencies andstimulus arrays within the operant chambers, are written using theMed-PC programming language for DOS.

The reinforcing effects of cocaine are assessed in a model thatquantitates the number of injections taken by rats under conditions inwhich intravenous administration is contingent upon a response made bythe animal (Mets et al., supra, 1998). The rats are trained in theoperant conditioning chambers to press a lever in order to gain accessto 0.5 ml of a sweetened milk solution. After the rats have acquired thelever-press response on a fixed-ratio 1 (FR1) schedule of reinforcement,the response requirements are successively increased to an FR5 schedule.When the rats display stable rates of milk-maintained responding overthree consecutive days on this schedule (less than 10% variability inreinforcer deliveries over the one-hour session) a catheter issurgically introduced in the left internal jugular vein and the rats aregiven a minimum of two days to recover from surgery.

On the first operant training session following surgery, rats areallowed to respond on the lever, in a one-hour session, for thesimultaneous 5-second delivery of both milk and an intravenous bolus ofcocaine (0.125 mg/kg/injection). The milk is then removed from thechamber and for the next three days, the rats are given access to one ofthree doses of cocaine (0.125, 0.25, or 0.5 mg/kg/injection) for onehour each, in self-administration sessions six hours in duration. Thus,the rats are allowed access to each dose twice per session and the dosesare presented in repeated ascending order (i.e., 0.125, 0.25, 0.5,0.125, 0.25, 0.5 mg/kg/injection). Within each one-hour longdose-component, the original FR5 schedule with a 10-second timeout isretained. In addition, 10-minute timeout periods are instituted aftereach dose component in an attempt to minimize carryover effects acrossthe individual one-hour sessions.

When the rats display consistent cocaine self-administration (over 160injections per six-hour session with less than 15% variability) overthree consecutive days, they are placed on a schedule in which smallerdoses, as well as saline, are available during single daily sessions.Each session is divided into two components, with saline and three dosesof cocaine available in each component. The first component of eachsession provides access to a series of low doses (0–0.0625mg/kg/injection) while the second component provides access to a widerrange of doses (0–0.5 mg/kg/injection).

After the rates of cocaine self-administration are stabilized the ratsare divided between six groups and each group (n=6 rats) is given 0.35,1.76, or 11.8 mg/kg of either wild-type butyrylcholinesterase, theoptimized butyrylcholinesterase variant or an equivalent volume ofsaline 30 minutes prior to the beginning of the dailyself-administration sessions. The effects of the pretreatment aremonitored for several days until the cocaine self-administrationbehavior of the rat returns to baseline.

Using a fixed ratio (FR) schedule, the number of injections is limitedonly by the duration of the session and consequently, the number ofinjections is used as the dependent variable to compare the potency ofoptimized butyrylcholinesterase with wild-type butyrylcholinesterase.Following administration of varying concentrations of wild-typebutyrylcholinesterase or the optimized butyrylcholinesterase variant,the dose response curves are analyzed using a mixed factor MANOVA. Thebutyrylcholinesterase concentration (0.35, 1.76, or 11.8 mg/kg) isloaded as the between-subjects factor and the cocaine dose (0, 0.015,0.03, 0.06, 0.125, 0.25, 0.5 mg/kg/injection) is loaded as thewithin-subjects factor. All individual comparisons acrossbutyrylcholinesterase treatment groups at individual cocaine doses usethe Tukey HSD post-hoc procedure (see Gravetter, F. J. and Wallnau, L.B., Statistics for the Behavioural Sciences (5th ed., 2000, WadsworthPubl., Belmont, Calif.)) and the criterion for statistical significanceis set at p < 0.05. At higher butyrylcholinesterase doses (11.8 mg/kg),the number of injections taken by the rats is expected to be lower thanthe untreated (saline) control group. Furthermore, rats treated with thebutyrylcholinesterase variant displaying enhanced cocaine hydrolysis areexpected to reduce their number of injections at a smaller dose (0.35mg/kg) than the animals treated with the wild-typebutyrylcholinesterase.

Drug discrimination is relevant to the subjective effect of cocaine inclinical situations and antagonism of cocaine discrimination followingpretreatment is considered clear evidence of therapeutic potential(Holtzman, Modern Methods in Pharmacology, Testing and Evaluation ofDrug Abuse, Wiley-Liss Inc., New York, (1990); Spealman, NIDA Res. Mon.119: 175–179 (1992)). The most frequently used procedure to establishand evaluate the discriminative stimulus effect of drugs is to trainanimals in a controlled operant procedure to use the injected drug as astimulus to control distribution of responding on two levers.Dose-effect curves consisting of distribution of the responses on the“drug-associated” lever as a function of drug dose are easily generated.These cocaine dose-effect curves can be altered by the administration ofa competitive antagonist. The amount of the shift of the curve and timerequired for the original sensitivity of the animal to cocaine to returnare useful data for evaluating the potential therapeutic use ofwild-type butyrylcholinesterase and the optimized variant. Thediscriminative stimulus effects of cocaine in rat models have been usedto evaluate the therapeutic potential of dopamine reuptake inhibitors,as well as agonists and antagonists to the dopamine receptors (Witkin etal., J. Pharmacol. Exp. Ther. 257: 706–713 (1989); Kantak et al, J.Pharmacol. Exp. Ther. 274: 657–665(1995); Barret and Appel,Psychopharmacology 99: 13–16 (1989); Callahan et al., Psychopharmacology103: 50–55(1991)).

A multiple trial procedure for training and testing cocaine as adiscriminative stimulus is used to evaluate the potency ofbutyrylcholinesterase in rats as previously described in Bertalmio etal. J. Pharmacol. Methods 7: 289–299 (1982) and Schecter, Eur. J.Pharmacol. 326: 113–118 (1997). A dose-response curve for cocaine isobtained in a single session in the presence of butyrylcholinesterase orthe optimized butyrylcholinesterase variant. Subsequently, the recoveryof the rat's original sensitivity to cocaine is tracked on atwice-weekly basis to assess the duration of action of thebutyrylcholinesterase.

The rats are deprived to 80% of their free-feeding weight at thebeginning of the experiment in order to train them in thefood-reinforced operant procedure. Each rat is placed in an operantconditioning chamber equipped with two light stimuli and two retractablelevers, one on either side of a milk delivery system and trained topress on one of the levers to receive access to 0.5 ml of sweetenedcondensed milk. Once the rats have learned to respond on this lever, amultiple-trials procedure is initiated. Each session consists of 6trials with each trial lasting 15 minutes. The first 10 minutes of eachtrial are a blackout period, during which no lights are on andresponding has no consequence. This 10-minute period allows for drugabsorption in the subsequent testing phases of the study. The last 5minutes of each trial are a milk-reinforced period (FR5). Once the ratsrespond consistently and rapidly during the 5-minute response period(signaling period), cocaine is introduced into the procedure.

Initially, 10 mg/kg cocaine is given 10 minutes prior to the beginningof three of six weekly sessions. During these sessions, the“non-cocaine” lever (saline) previously extended is retracted and theother, “cocaine-associated,” lever is extended on the other side of themilk delivery cup. Responses (initially only a single response;eventually five responses) on this second lever result in milkpresentation if cocaine was administered prior to the session. The ratsare being trained to respond on the second lever if they detect theinteroceptive effects of the administered cocaine. Because cocaine'sinteroceptive effects are not believed to extend beyond 30 minutes, thesessions following cocaine administration lasts for only two trials (15minutes each). At this juncture the rats do not receive a cocaineinjection on three days of the week and on those days they arereinforced with milk (FR5) for responding on the available non-cocainelever during the signaling periods of six trials. On the remaining threedays of the week, the rats are given 10 mg/kg cocaine before thebeginning of the session and are reinforced for responding on theavailable cocaine lever during the signaling periods on each of twotrials.

Subsequently, each daily session is initiated with one to four trialswithout cocaine administration, followed by the administration of 10mg/kg cocaine. Thus, each session ends with two trials in whichresponding on the cocaine-appropriate lever is required for fooddelivery. Although only the “correct” levers are extended during thisphase, the critical step of making both levers available during theentire session is taken as soon as the animals learn to switch from thenon-cocaine to the cocaine lever within daily sessions. Subsequently,each session begins with a 10-minute blackout period followed bypresentation of both levers for five minutes. During the first 1 to 4trials of a daily session, no cocaine is given, and 5 consecutiveresponses on the non-cocaine lever result in food during this 5-minuteperiod. If the rat switches from one lever to the other or responds onthe incorrect lever, he does not get reinforced and both levers areretracted for 10 seconds, at which time the levers are presented againand the trial restarted. At the start of the second, third, or fourthtrial, 10 mg/kg cocaine are given and the rat is returned to the testbox. When the light is illuminated and the levers presented on the nexttwo trials, five consecutive responses on the cocaine lever are requiredfor milk presentation to demonstrate that the rats are learning toswitch their responding from the non-cocaine lever to the cocaine leverusing the interoceptive effects of cocaine as a cue to tell them whichlever is correct on a given trial.

A cocaine dose-effect curve is obtained as soon as the rats meetcriterion of 80% correct lever selection on three consecutive sessions.On the first trial of a test session, saline is given. On subsequenttrials, 0.1, 0.3, 1.0, 3.2, and 10 mg/kg cocaine is administered, eachat the start of the 10 minute blackout that begins each trial. Duringthese test trials, five consecutive responses on either lever result inmilk presentation, but switching from one lever to the other prior tocompletion of an FR results in lever retraction for 10 seconds. It isanticipated that animals begin this session with responses on thenon-cocaine lever and gradually increase the percent of responses madeon the cocaine lever until all responses are made on that lever. Thus, adose-response curve of lever selection versus dose of cocaineadministered is established during each test session.

Once cocaine has been established as a discriminative stimulus, the ratsare placed in separate groups (n=6 per group) that receive 0.35, 1.76,or 11.8 mg/kg of either wild-type butyrylcholinesterase or the optimizedvariant. The discriminative stimulus effects of cocaine is determined 30minutes following enzyme administration and daily afterwards untilsensitivity to cocaine is re-established. On the initial test sessionfollowing administration of butyrylcholinesterase, larger doses ofcocaine are given if there is no selection of the cocaine leverfollowing any of the smaller test doses. Doses as large as 100 mg/kgcocaine are given if the animals fail to select the cocaine-appropriatelever following administration of 10 or 32 mg/kg cocaine. Becausedose-response curves to cocaine can be obtained in a single session,this protocol provides information on the relative ability of the twotypes of butyrylcholinesterase to decrease the potency of cocaine as adiscriminative stimulus, which is a relevant aspect of its abuseliability. The butyrylcholinesterase variant displaying enhanced cocainehydrolysis activity in vitro is more potent.

Throughout this application various publications have been referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1. A butyrylcholinesterase variant comprising an amino acid sequencecomprising a sequence of amino acid residues 1 or 29 through 602,inclusive, of the amino acid sequence shown as SEQ ID NO:2, wherein saidvariant has tryptophan at amino acid position
 356. 2. Thebutyrylcholinesterase variant of claim 1, having a 15-fold increase incocaine hydrolysis activity compared to human butyrylcholinesterase.